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Premotoneuronal inputs to early developing trigeminal motoneurons
Journal of Oral Biosciences ∎ (∎∎∎∎) ∎∎∎–∎∎∎
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Original Article
Premotoneuronal inputs to early developing trigeminal motoneurons Keishi Matsuda a,c, Shiro Nakamura a,n, Mutsumi Nonaka b, Ayako Mochizuki a, Kiyomi Nakayama a, Takehiko Iijima b, Atsuro Yokoyama c, Makoto Funahashi d, Tomio Inoue a a
Department of Oral Physiology, Showa University School of Dentistry, 1-5-8 Hatanodai, Shinagawa-ku, Tokyo 142-8555, Japan Department of Perioperative Medicine, Division of Anesthesiology, Showa University School of Dentistry, 1-5-8 Hatanodai, Shinagawa-ku, Tokyo 142-8555, Japan c Department of Oral Functional Prosthodontics, Graduate School of Dental Medicine, Hokkaido University, Kita 13 Nishi 7, Kita-ku, Sapporo 060-8586, Japan d Department of Oral Physiology, Graduate School of Dental Medicine, Hokkaido University, Kita 13 Nishi 7, Kita-ku, Sapporo 060-8586, Japan b
art ic l e i nf o
a B S T R AC T
Article history: Received 23 August 2016 Received in revised form 13 December 2016 Accepted 18 December 2016
Objectives: We previously reported that masseter motoneurons (MMNs) and digastric motoneurons (DMNs) in postnatal day (P) 1–5 rats receive convergent inputs from the lateral (l-) and medial (m-) supratrigeminal regions (SupV), intertrigeminal region (IntV), and dorsal region of the principal sensory trigeminal nucleus (PrV). The l-SupV sends burst inputs predominantly to the MMNs. We compared the synaptic inputs to P9–12 rat MMNs and DMNs with those found in the previous study involving P1–5 rats. Methods: We performed whole-cell recordings and laser photolysis of caged glutamate in the MMNs and DMNs of P9–12 rats. Results: Similar to P1–5 rats, the photostimulation of multiple regions within the l-SupV, m-SupV, IntV, and dorsal PrV, induced postsynaptic currents (PSCs) in both P9–12 MMNs and DMNs. Photostimulation induced predominantly low-frequency PSCs in both P9–12 motoneurons, whereas l-SupV photostimulation predominantly induced burst PSCs in P1–5 rats. However, when the caged glutamate concentration was doubled, l-SupV photostimulation evoked burst PSCs in all P9–12 MMNs. Furthermore, l-SupV and m-SupV photostimulation evoked burst or low-frequency PSCs at significantly higher rates in the MMNs compared to in the DMNs. Conclusions: These results suggested that, similar to P1–5 motoneurons, both P9–12 motoneurons received convergent inputs from the SupV, IntV, and PrV; however, the input–output gains of some of the premotor neurons decreased. These synaptic input changes may contribute to the proper development of chewing. & 2017 Japanese Association for Oral Biology. Published by Elsevier B.V. All rights reserved.
Keywords: Laser photolysis Caged glutamate Supratrigeminal region Synaptic input Patch clamp
1. Introduction Feeding behavior changes drastically during the postnatal period in mammals. Suckling is first performed, following which chewing develops. The muscles that close the jaw show little activity during suckling; however, these muscles require high levels of activity in animals chewing hard or tough food [1–3]. Thus, the motor commands sent to the masticatory muscles from the neural circuits that control suckling and chewing likely change as the teeth and orofacial musculoskeletal system develop. The last-order Abbreviations: ACSF, artificial cerebrospinal fluid; AP, action potential; DMN, digastric motoneurons; dRt, reticular formation dorsal to the PrV; IntV, intertrigeminal region; l, lateral; m, medial; MMN, masseter motoneurons; MoV, trigeminal motor nucleus; P, postnatal; PrV, principal sensory trigeminal nucleus; PSC, postsynaptic current; SupV, supratrigeminal region n Corresponding author. E-mail address: [email protected] (S. Nakamura).
premotor neurons that transmit such motor commands to the trigeminal motoneurons play an important role in controlling masticatory muscle activity [4,5]. The supratrigeminal (SupV) and intertrigeminal (IntV) regions, the dorsal regions of the principal sensory trigeminal nucleus (PrV), and the reticular formation dorsal to the PrV (dRt) that surround the trigeminal motor nucleus (MoV) contain premotor neurons that innervate the motoneurons of the muscles that open and close the jaw [6,7]. Furthermore, the premotor neurons in these regions that target the MoV receive afferent inputs from a number of regions, such as the orofacial structures [8–10], central pattern generator for mastication [4,11], cerebral cortex [12], amygdala [13], and lateral hypothalamus [14]. Therefore, these premotor neurons might transmit the motor commands for suckling and/or chewing. We previously reported that, in postnatal day (P) 1–5 neonatal rats, both single masseter motoneurons (MMNs) and digastric motoneurons (DMNs) receive convergent glutamatergic inputs from the SupV, IntV, PrV, and dRt,
http://dx.doi.org/10.1016/j.job.2017.01.003 1349-0079/& 2017 Japanese Association for Oral Biology. Published by Elsevier B.V. All rights reserved.
Please cite this article as: Matsuda K, et al. Premotoneuronal inputs to early developing trigeminal motoneurons. J Oral Biosci (2017), http://dx.doi.org/10.1016/j.job.2017.01.003i
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and that the lateral SupV sends burst inputs predominantly to the MMNs [15]. However, it is uncertain whether single motoneurons in juvenile (P9–12) rats receive convergent inputs from premotor neurons in the SupV, IntV, PrV, and dRt. Furthermore, it is unclear whether the premotor neurons in the lateral SupV of P9–12 rats send burst inputs predominantly to the MMNs. In this study, we investigated the synaptic inputs to MMNs and DMNs from the SupV, IntV, PrV, and dRt in P9–12 rats using the laser photolysis of caged glutamate. This allowed for the systematic stimulation of multiple confined regions, and simultaneous whole-cell recordings of motoneurons that were identified by a fluorescent dye.
2. Materials and methods 2.1. Slice preparation The experiments were performed on coronal slices of the brainstem from P9–12 Wistar rats (n ¼ 59). The slices were prepared as described in the Supplementary materials. 2.2. Retrograde labeling of jaw-closing and jaw-opening motoneurons In order to discriminate between the motoneurons innervating the muscles that open or close the jaw, we used the fluorescence labeling technique [16,17] as described in the Supplementary materials. 2.3. Patch-clamp recordings Whole-cell patch-clamp recordings were performed in the SupV neurons and the jaw-closing and jaw-opening motoneurons according to the procedures described in the Supplementary materials. 2.4. Photostimulation We systematically stimulated various sites in the slices while simultaneously performing whole-cell recordings in the SupV neurons or motoneurons. Caged glutamate was subjected to laser photolysis by a 365-nm nitrogen-pulsed Micropoint laser system equipped with galvanometer-based steering lenses (Photonic Instruments, Inc., St. Charles, IL) [cf. 15]. The laser beam was positioned by steering the lenses with the MetaMorph software (Molecular Devices, LLC, Sunnyvale, CA). After using a 40X water immersion objective to establish the whole-cell recording configuration in an MMN, DMN, or SupV neuron, we changed the 40 objective to an Olympus 4 (0.28 NA) objective. Furthermore, 4-methoxy-7-nitroindolinyl-caged L-glutamate (MNI glutamate; Tocris Bioscience, Bristol, UK) was added to the recirculating artificial cerebrospinal fluid (ACSF) to a final concentration of 300 mM in 25 mL of ACSF. In some experiments, the final MNI glutamate concentration was increased to 600 mM. All the photostimulation experiments began at least 10 min after the addition of MNI glutamate. The laser beam was focused onto an area of approximately 10 mm in diameter on the brainstem slice using the 4 objective. Single laser-beam pulses (2–6 ns) were delivered to the center of each site to trigger the focal photolysis of MNI glutamate. The photostimulation strength was always set at 70 (a.u.) in the MetaMorph software, which was the same setting used to stimulate the P1–5 rats in our previous experiment [15]. Each site was stimulated 4–8 times. The response pattern (burst PSC, low-frequency PSC, or no response) shown by over 60% of the responses across 4–8 trials was considered the representative response pattern of each sample at each stimulation site. Responses of more
than 25 pA were considered as effectively evoking postsynaptic responses. To determine the spatial resolution of the photostimulation, a single-pulsed laser beam was delivered at 5-s intervals to 100 different sites that surrounded the recording sites of the SupV neurons. The 100 sites were arranged in a 10 10 array with 40mm spaces between adjacent rows and columns (Supplementary Fig. 1A). As the brainstem is larger in P9–12 animals than in P1–5 animals, and the laser beam could only be positioned within an area of about 780 1200 mm2 in our system, we could not stimulate all the areas in the SupV, IntV, and PrV of the P9–12 animals in singlemotoneuron recording sessions. Thus, we stimulated 76 different sites in 36 slices at 2-s intervals. The photostimulation sites adjacent to the MoV, covering the SupV, IntV, and the medial border of the PrV, were arranged in an L-shape, with distances of 120 mm and 78 mm between the neighboring sites in the dorsal-ventral and medial-lateral orientations. The boundary of the MoV was identified using the 40 objective. We assigned the areas of the m-SupV, l-SupV, IntV, and PrV based on histological observations made from Nissl-stained slices. In 9 different slices, photostimulation was delivered to 40 sites in the more lateral regions, including the medial PrV. These 40 sites were arranged in an I-shape with distances of 120 mm and 78 mm between the neighboring sites in the dorsal-ventral and medial-lateral orientations (Fig. 4A). 2.5. Histological procedure The histological identification of the photostimulation sites was performed as described in the Supplementary materials. 2.6. Statistics The data are presented as the mean 7 SEM, except when specifically indicated. The data were compared within and between groups using the nonparametric Mann-Whitney U-test. Results with probability (p) values less than 0.05 were considered statistically significant. When the comparisons were performed among 4 groups, results with p values less than 0.0083 (0.05/6) were considered statistically significant. The statistical analyses were performed using the SPSS Statistics 17.0 (SPSS Japan, Tokyo, Japan) software.
3. Results 3.1. Spatial resolution of the photostimulation in P9–12 rats In order to quantify the spatial resolution of the action potentials of putative premotor neurons by a single laser-beam pulse at P9–12 rats, we examined the effects of a single stimulation pulse on the action potentials (APs) of individual putative SupV premotor neurons with a method similar to that used to examine P1– 5 rats [15]. Photostimulation applied close to the soma or proximal dendrites evoked APs (Supplementary Fig. 1A, 1D). Stimulation at some sites evoked bursts of APs at frequencies greater than 50 Hz (Supplementary Fig. 1Dc, e, 1Ec, e). For the 19 SupV neuron recordings, sites in which stimulation evoked at least one AP were located within a 103 7 43.4 mm (mean 7 SD, median: 89.4 mm; range: 40–179 mm) radius from the tip of the patch pipette, which corresponded to the location of the soma (Supplementary Fig. 1De). This value was similar to the 126 7 22.7-mm radius for P1–5 rats [15]. Thus, if the two effective photostimulation sites that evoked postsynaptic responses in a motoneuron were separated by more than twice the radius, the
Please cite this article as: Matsuda K, et al. Premotoneuronal inputs to early developing trigeminal motoneurons. J Oral Biosci (2017), http://dx.doi.org/10.1016/j.job.2017.01.003i
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Fig. 1. Mapping of the synaptic connections between premotor neurons in the supratrigeminal region (SupV) and intertrigeminal region (IntV) and a masseter motoneuron (MMN) investigated with the laser photolysis of MNI glutamate (A) Low-magnification video image of a coronal slice preparation from a postnatal-day (P) 10 rat obtained during recording overlaid by the 76 stimulation sites arranged in an L shape (upper panel) and a photomicrograph of the corresponding Nissl-stained slice (lower panel). The soma of the recorded neuron is indicated by an asterisk. Scale bar ¼ 500 mm. (B) High-magnification translucent (upper panel) and epifluorescent (lower panel) photomicrographs of the recorded MMN (indicated by arrowheads). Scale bar ¼ 50 mm. (C) Current responses corresponding to the stimulation grid shown in (A). The a, b, c, d, and e labels denote the responses that were evoked by the stimulation of the positions labeled with the same letters in (A). (D) Expanded traces of the a, b, c, d, and e response recordings shown in (C). The latencies of the currents in the a, b, c, d and e were 7.7, 9.8, 39.3, 18.9, and 0.54 ms, respectively. The slowly rising inward current in ‘e’ was evoked at a much shorter latency by stimulation of sites near the trigeminal motor nucleus (MoV), such as the ‘e’ recording shown in (C) and (D). This response was most likely evoked by the direct stimulation of the recorded MMN by uncaged glutamate [16]. This MMN exhibited the postsynaptic currents (PSCs) in response to the photostimulation of 3 different regions (l-SupV, m-SupV, and IntV). MoV, trigeminal motor nucleus; l-SupV and m-SupV, lateral and medial supratrigeminal regions, respectively; IntV, intertrigeminal region; PrV, principal sensory trigeminal nucleus; dRt, reticular formation dorsal to the PrV; Lat, lateral; Dor, dorsal.
two effective sites were considered representations of two different premotor neurons that innervated the recorded motoneuron. To avoid overestimation of the number of regions that send inputs to single motoneurons, we adopted a 252-mm radius criterion (i.e., two times 126 mm) rather than a 206-mm radius (i.e., two times 103 mm) in the evaluations of the number of inputs. 3.2. Postsynaptic responses in P9–12 motoneurons As photostimulation of the SupV and IntV evoked postsynaptic currents (PSCs) in the MMNs and DMNs in P1–5 rats [15], we examined the effects of photostimulation of the SupV and IntV on 27 MMNs and 20 DMNs in P9–12 rats. Rapidly rising inward PSCs were evoked at latencies of 5.4–40.0 ms in MMNs and DMNs when the lateral SupV (l-SupV), medial SupV (m-SupV), and IntV were stimulated (Figs. 1A–D and 2A–D). Slowly rising inward currents that were evoked at much shorter latencies of approximately 0.5 ms (below 1 ms) by stimulation applied to the sites (indicated by “e” in Fig. 1A and “d” in Fig. 2A) most likely result from the direct stimulation of the recorded motoneurons by uncaged glutamate (Fig. 1De, 2Dd) [16]. In contrast to results from P1–5 animals, lowfrequency PSCs (single, double, or multiple current responses at frequencies below 50 Hz; Fig. 1Cc, d, 1Dc, d) were the predominant responses to stimulation in the MMNs. Burst PSCs, which were defined as multiple currents comprising more than 3 sharp inward currents at high frequencies (450 Hz; Fig. 1Ca, b, 1Da, b), were less frequent. We evaluated the input profiles to single motoneurons from the SupV and IntV. Since we have previously demonstrated that the differences in electrophysiological and histological properties between the l-SupV and m-SupV [15,18], we distinguished the SupV into l-SupV and m-SupV for the evaluation
of input profiles. The upper panel of Fig. 3 shows the mean induction profiles of the burst and low-frequency PSCs that were evoked in the 27 MMNs. Photostimulation of 12 and 15 spots that approximately correspond to the l-SupV and m-SupV (outlined by dotted and dashed squares, respectively, in Fig. 3) evoked more low-frequency PSCs than burst PSCs in the MMNs (l-SupV: U ¼ 144.0, p o 0.0016; m-SupV: U ¼ 72.5, p ¼ 0.0016). Similar to the MMNs, low-frequency PSCs were evoked more often than burst PSCs (l-SupV: U ¼ 37.0, p o 0.0016; m-SupV: U ¼ 61.5, p o 0.0016; Figs. 2C, 2D, 3A) in the DMNs by l-SupV and m-SupV stimulation. However, the incidence of low-frequency PSCs in response to m-SupV stimulation was significantly higher in the MMNs compared to that in the DMNs (U ¼ 138.5, p o 0.0083; Fig. 3A, lower panel). There was no significant difference in the incidence of low-frequency PSCs in response to l-SupV stimulation between the MMNs and DMNs (U ¼ 197.5, p ¼ 0.114). Although burst PSCs were less frequent, they were evoked more in the MMNs compared with the DMNs by l-SupV and m-SupV stimulation (l-SupV: U ¼ 136.0, p ¼ 0.0016; m-SupV: U ¼ 152.5, p o 0.0083; Fig. 3A). The induction rates of either burst or lowfrequency PSCs in response to l-SupV and m-SupV stimulation were significantly higher in the MMNs compared with the DMNs (l-SupV: U ¼ 131.0, p o 0.01; m-SupV: U ¼ 116.5, p o 0.01; Fig. 3B). The profiles of the inputs to the motoneurons in P9–12 rats were similar to those in P1–5 rats. For many MMNs and DMNs, PSCs were evoked in multiple sites that were separated by more than 252 mm (Fig. 1Ca, b, c; Fig. 2Ca, b, c). We found that most of the MMNs (89%, 24/27) and DMNs (75%, 15/20) exhibited PSCs in response to photostimulation of multiple regions (Supplementary table 1).
Please cite this article as: Matsuda K, et al. Premotoneuronal inputs to early developing trigeminal motoneurons. J Oral Biosci (2017), http://dx.doi.org/10.1016/j.job.2017.01.003i
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Fig. 2. Mapping of the synaptic connectivity between the premotor neurons and a digastric motoneuron (DMN) (A) Low-magnification video image of a coronal slice preparation from a P10 rat obtained during recording overlaid by the 76 stimulation sites arranged in an L shape (upper panel) and a photomicrograph of the corresponding Nissl-stained slice (lower panel). The soma of the recorded neuron is indicated by an asterisk. Scale bar ¼ 500 mm. (B) High-magnification translucent (upper panel) and epifluorescent (lower panel) photomicrographs of the recorded DMN (indicated by arrowheads). Scale bar ¼ 50 mm. (C) Current responses corresponding to the stimulation grid shown in (A). The a, b, c, and d labels denote the postsynaptic currents evoked by the stimulation of the positions labeled with the same letters in (A). (D) Expanded traces of the a, b, c, and d response recordings shown in (C). The latencies of the currents in the a, b, c, and d were 7.2, 12.9, 17.3, and 0.53 ms, respectively. This DMN showed PSCs in response to the photostimulation of 3 different regions (l-SupV, m-SupV, and IntV). MoV, trigeminal motor nucleus; l-SupV and m-SupV, lateral and medial supratrigeminal regions, respectively; IntV, intertrigeminal region; PrV, principal sensory trigeminal nucleus; dRt, reticular formation dorsal to the PrV; Lat, lateral; Dor, dorsal.
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Fig. 3. Induction profile of the burst or low-frequency postsynaptic currents (PSCs) that were evoked in masseter motoneurons (MMNs) and digastric motoneurons (DMNs) by photostimulation of the SupV and IntV in P9–12 rats (A) Pseudocolor maps of the incidence of burst or low-frequency PSCs in the MMNs (upper panel) and DMNs (lower panel) for each site. *p o 0.0083; **p o 0.0016. (B) Pseudocolor maps of the incidence of either burst or low-frequency PSCs in the MMNs (upper panel) and DMNs (lower panel) for each site. ‡p o 0.01. The dotted and dashed squares in (A) and (B) outline the l-SupV and m-SupV, respectively. l-SupV and m-SupV, lateral and medial supratrigeminal regions, respectively; IntV, intertrigeminal region; Lat, lateral; Dor, dorsal.
In another set of slices, photostimulation was applied to the PrV, and the responses of 9 MMNs and 8 DMNs were examined. Photostimulation of the dorsal PrV evoked low-frequency PSCs in 6 MMNs (66%, Figs. 4C, 4D) and 4 DMNs (50%, Figs. 5C, 5D), whereas burst PSCs were evoked in only 2 MMNs (22%, Fig. 4Ca, 4 Da) and 1 DMN (13%, data not shown). For both MMNs and DMNs, the incidence of burst PSCs evoked by PrV stimulation did
not differ significantly from that of low-frequency PSCs (Fig. 6). When the PrV was divided into the ventral and dorsal parts for comparison, no significant differences in the induction rate were detected between the MMNs and DMNs and/or between the burst and low-frequency PSCs. (Fig. 6). To determine the existence of inhibitory components in the photostimulation-evoked PSCs of the P9–12 motoneurons, we
Please cite this article as: Matsuda K, et al. Premotoneuronal inputs to early developing trigeminal motoneurons. J Oral Biosci (2017), http://dx.doi.org/10.1016/j.job.2017.01.003i
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Fig. 4. Mapping of the synaptic connectivity between premotor neurons in the PrV and a masseter motoneuron (MMN) (A) Low-magnification video image of a coronal slice preparation from a P11 rat obtained during recording overlaid by the 40 stimulation sites arranged in an I shape (upper panel) and a photomicrograph of the corresponding Nissl-stained slice (lower panel). The soma of the recorded neuron is indicated by an asterisk. Scale bar ¼ 500 mm. (B) High-magnification translucent (upper panel) and epifluorescent (lower panel) photomicrographs of the recorded MMN (indicated by arrowheads). Scale bar ¼ 50 mm. (C) Current responses corresponding to the stimulation grid shown in (A). The a and b labels denote the postsynaptic currents that were evoked by stimulation of the positions labeled by the same letters in (A). (D) Expanded traces of the a and b response recordings shown in (C). The latencies of the currents in a and b were 5.7 and 21.2 ms, respectively. MoV, trigeminal motor nucleus; PrV, principal sensory trigeminal nucleus; Lat, lateral; Dor, dorsal.
recorded the PSCs evoked by photostimulation of the SupV and IntV at a holding potential of 20 mV in P9–12 MMNs (n ¼ 7). No outward PSCs were evoked by photostimulation in any of the 7 MMNs tested in P9–12 rats. 3.3. High-intensity photostimulation The low incidence of burst PSCs in P9–12 rats might have been due to decreased excitability in the premotor neurons that generate burst PSCs in trigeminal motoneurons during postnatal development. Therefore, we examined the effects of higher concentrations of MNI glutamate on PSCs in MMNs (Fig. 7A–E). Photostimulation of the l-SupV with the application of 600-mM MNI glutamate evoked burst PSCs in all 6 tested MMNs, while photostimulation at the same sites with the application of 300-mM MNI glutamate evoked low-frequency PSCs in the same MMNs (Fig. 7Ca, b, 7Da, b, 7Ea, b).
4. Discussion In the present study, we showed that low-frequency PSCs were the predominant responses in both MMNs and DMNs to photostimulation of the l-SupV and m-SupV in P9–12 rats. We have recently shown that more than one-third of the SupV premotor neurons in P1–6 rats fire at rates over 50 Hz [18]. Similarly, we have shown in both P1–5 [15] and P9–12 (present study) rats that photostimulation of the cell bodies of SupV neurons increases the firing rates of some neurons to over 50 Hz. Therefore, some
premotor neurons in the SupV probably evoke burst PSCs in trigeminal motoneurons. Furthermore, when we increased the MNI glutamate concentrations from 300 mM to 600 mM in P9–12 rats, the PSCs evoked in the MMNs by l-SupV photostimulation changed from a low-frequency pattern to a burst pattern. If the premotor neurons that innervate the trigeminal motoneurons increase in size during development like trigeminal motoneurons do [19], then the input resistances of the premotor neurons probably decrease. Thus, these results suggest that, in P9–12 rats, the uncaged glutamate induces only small depolarizations and low-frequency firing in the majority of premotor neurons in the SupV and PrV, whereas the same amount of uncaged glutamate induces large depolarizations and bursting firing patterns in many premotor neurons in the SupV and PrV in P1–5 rats. These changes in the input–output gains of the premotor neurons during photostimulation are, at least in part, responsible for the low incidence of burst PSCs in the MMNs of P9–12 rats. However, we cannot rule out the possibility that some of the premotor neurons that generate burst spikes and burst PSCs in the motoneurons of neonates may change their firing patterns during postnatal development to single spikes. We did not determine whether the evoked PSCs in the MMNs and DMNs of P9–12 rats were excitatory or inhibitory when the MNI glutamate was uncaged. Although electrical stimulation of the SupV in P1–12 rats evokes glycinergic and GABAergic PSCs in MMNs and DMNs, as well as more potent glutamatergic PSCs [16], the application of GABAA and glycine receptor antagonists in P1–5 rats did not substantially alter the induction profiles of evoked PSCs in MMNs and DMNs by photostimulation with MNI
Please cite this article as: Matsuda K, et al. Premotoneuronal inputs to early developing trigeminal motoneurons. J Oral Biosci (2017), http://dx.doi.org/10.1016/j.job.2017.01.003i
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Fig. 5. Mapping of the synaptic connectivity between premotor neurons in the PrV and a digastric motoneuron (DMN) (A) Low-magnification video image of a coronal slice preparation from a P11 rat obtained during recording overlaid by the 40 stimulation sites arranged in an I shape (upper panel) and a photomicrograph of the corresponding Nissl-stained slice (lower panel). The soma of the recorded neuron is indicated by an asterisk. Scale bar ¼ 500 mm. (B) High-magnification translucent (upper panel) and epifluorescent (lower panel) images of the recorded DMN (indicated by arrowheads). Scale bar ¼ 50 mm. (C) Current responses corresponding to the stimulation grid shown in (A). The a and b labels denote the postsynaptic currents that were evoked by the stimulation of the positions labeled by the same letters in (A). (D) Expanded traces of a and b response recordings shown in (C). The latencies of the currents in a and b were 15.5 and 17.1 ms, respectively. MoV, trigeminal motor nucleus; PrV, principal sensory trigeminal nucleus; Lat, lateral; Dor, dorsal.
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0
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DMN n=8 Lat 200 µm Fig. 6. Induction profile of the burst or low-frequency postsynaptic currents were evoked in the masseter motoneurons (MMNs) and digastric motoneurons (DMNs) by photostimulation of the PrV in P9–12 rats (A, B) Pseudocolor maps of the incidence of burst PSCs [left panel in (A)], low-frequency [right panel in (A)], and both (B) PSCs in the MMNs (upper panel) and DMNs (lower panel) for each site. PrV, principal sensory trigeminal nucleus; Lat, lateral; Dor, dorsal.
glutamate [15]. Furthermore, we failed to evoke outward PSCs in any of the 7 MMNs tested in P9–12 rats by the uncaging of MNI glutamate (holding potential of 20 mV, patch-pipette internal solution with 0.4 mM Cl ). However, Fino et al. [20] have reported that MNI glutamate strongly blocks GABAergic responses. Therefore, in the present study, the MNI glutamate might have blocked glycinergic and GABAergic transmission to the MMNs and DMNs, and photostimulation might have evoked excitatory PSCs. In addition, we demonstrated that the photostimulation of either l-SupV or m-SupV significantly evoked PSCs in MMNs at higher rates than in DMNs in the P9–12 rats that were about to initiate chewing behavior. The activities of the jaw-closing muscles are more easily facilitated by the physical properties of food compared to the activities of the jaw-opening muscles through the activation of periodontal and spindle afferents [2,3,21]. The SupV receives periodontal and spindle afferent inputs [22–25], and the premotor neurons that target the MoV and that are rhythmically active during the jaw-closing phase of cortically induced fictive mastication have been identified in the SupV of anesthetized and immobilized rats [9]. Thus, we identified that the pattern of inputs from the l-SupV and m-SupV to the MMNs was dominant compared to the inputs to the DMNs. These results suggested that the premotor neurons in the SupV are responsible for increasing the activity of the jaw-closing muscles in response to periodontal and spindle afferent inputs during mastication. In addition, in contrast to P1–5, low-frequency PSCs in response to SupV photostimulation were predominant in the MMNs and DMNs in P9–12 rats, owing to, at least in part, developmental changes in the input-output gains of the premotor neurons. These developmental changes in
Please cite this article as: Matsuda K, et al. Premotoneuronal inputs to early developing trigeminal motoneurons. J Oral Biosci (2017), http://dx.doi.org/10.1016/j.job.2017.01.003i
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Fig. 7. The effects of photostimulation of the SupV by a high concentration of MNI glutamate on the postsynaptic currents (PSCs) evoked in the MMNs (A) Low-magnification video image of a coronal slice preparation from a P12 rat obtained during recording overlaid by the 40 stimulation sites arranged in an I shape. The soma of the recorded neuron is indicated by an asterisk. Scale bar ¼ 500 mm. (B) High-magnification translucent (upper panel) and epifluorescent (lower panel) photomicrographs of the recorded MMN (indicated by arrowheads). Scale bar ¼ 50 mm. (C, D) Current responses corresponding to the stimulation grid shown in (A) recorded during the application of 300 mM MNI glutamate (C) or 600 mM MNI glutamate (D). The a and b labels denote the PSCs evoked by the stimulation of positions labeled by the same letters in (A). (E) Expanded traces of a and b response recordings shown in (C) and (D) in response to the application of 300 mM MNI glutamate (upper panel) and 600 mM MNI glutamate (lower panel), respectively. Robust PSCs were evoked in this MMN when the l-SupV was stimulated by 600 mM MNI glutamate, while photostimulation of the same area by 300 mM MNI glutamate evoked low-frequency PSCs. The latencies of the currents at the application of 300 mM and 600 mM in a were 9.2 and 5.8 ms, respectively. The latencies of the currents at the application of 300 mM and 600 mM in b were 18.3 and 20.6 ms, respectively. MoV, trigeminal motor nucleus; SupV, supratrigeminal regions; Lat, lateral; Dor, dorsal.
the properties of synaptic inputs from the SupV to trigeminal motoneurons may contribute to the proper development of chewing.
5. Conclusions We demonstrated that both P9–12 rat MMNs and DMNs received convergent inputs from the SupV, IntV, and PrV, similar to P1–5 rats, and the input–output gains of some of the premotor neurons were likely to be lower in P9–12 rats. Furthermore, we found that the MMNs received a larger number of inputs from the SupV than the DMNs did in P9–12 rats. These changes in the properties of synaptic inputs from the premotor neurons to trigeminal motoneurons may contribute to the proper development of feeding behavior.
Conflicts of interest The authors have no conflicts of interest to declare.
Ethical approval All of the experiments were approved by the International Animal Research Committee of the Showa University in accordance with the Japanese Government Law No. 105 for the care and use of laboratory animals.
Acknowledgements This work was supported by the MEXT-Supported Program for the Strategic Research Foundation at Private Universities 2012– 2016 and 2014–2018, and JSPS KAKENHI Grants JP26293397, JP15K15687, and JP16K11488.
Appendix A. Supporting information Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.job.2017.01.003.
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Original Article
Premotoneuronal inputs to early developing trigeminal motoneurons Keishi Matsuda a,c, Shiro Nakamura a,n, Mutsumi Nonaka b, Ayako Mochizuki a, Kiyomi Nakayama a, Takehiko Iijima b, Atsuro Yokoyama c, Makoto Funahashi d, Tomio Inoue a a
Department of Oral Physiology, Showa University School of Dentistry, 1-5-8 Hatanodai, Shinagawa-ku, Tokyo 142-8555, Japan Department of Perioperative Medicine, Division of Anesthesiology, Showa University School of Dentistry, 1-5-8 Hatanodai, Shinagawa-ku, Tokyo 142-8555, Japan c Department of Oral Functional Prosthodontics, Graduate School of Dental Medicine, Hokkaido University, Kita 13 Nishi 7, Kita-ku, Sapporo 060-8586, Japan d Department of Oral Physiology, Graduate School of Dental Medicine, Hokkaido University, Kita 13 Nishi 7, Kita-ku, Sapporo 060-8586, Japan b
art ic l e i nf o
a B S T R AC T
Article history: Received 23 August 2016 Received in revised form 13 December 2016 Accepted 18 December 2016
Objectives: We previously reported that masseter motoneurons (MMNs) and digastric motoneurons (DMNs) in postnatal day (P) 1–5 rats receive convergent inputs from the lateral (l-) and medial (m-) supratrigeminal regions (SupV), intertrigeminal region (IntV), and dorsal region of the principal sensory trigeminal nucleus (PrV). The l-SupV sends burst inputs predominantly to the MMNs. We compared the synaptic inputs to P9–12 rat MMNs and DMNs with those found in the previous study involving P1–5 rats. Methods: We performed whole-cell recordings and laser photolysis of caged glutamate in the MMNs and DMNs of P9–12 rats. Results: Similar to P1–5 rats, the photostimulation of multiple regions within the l-SupV, m-SupV, IntV, and dorsal PrV, induced postsynaptic currents (PSCs) in both P9–12 MMNs and DMNs. Photostimulation induced predominantly low-frequency PSCs in both P9–12 motoneurons, whereas l-SupV photostimulation predominantly induced burst PSCs in P1–5 rats. However, when the caged glutamate concentration was doubled, l-SupV photostimulation evoked burst PSCs in all P9–12 MMNs. Furthermore, l-SupV and m-SupV photostimulation evoked burst or low-frequency PSCs at significantly higher rates in the MMNs compared to in the DMNs. Conclusions: These results suggested that, similar to P1–5 motoneurons, both P9–12 motoneurons received convergent inputs from the SupV, IntV, and PrV; however, the input–output gains of some of the premotor neurons decreased. These synaptic input changes may contribute to the proper development of chewing. & 2017 Japanese Association for Oral Biology. Published by Elsevier B.V. All rights reserved.
Keywords: Laser photolysis Caged glutamate Supratrigeminal region Synaptic input Patch clamp
1. Introduction Feeding behavior changes drastically during the postnatal period in mammals. Suckling is first performed, following which chewing develops. The muscles that close the jaw show little activity during suckling; however, these muscles require high levels of activity in animals chewing hard or tough food [1–3]. Thus, the motor commands sent to the masticatory muscles from the neural circuits that control suckling and chewing likely change as the teeth and orofacial musculoskeletal system develop. The last-order Abbreviations: ACSF, artificial cerebrospinal fluid; AP, action potential; DMN, digastric motoneurons; dRt, reticular formation dorsal to the PrV; IntV, intertrigeminal region; l, lateral; m, medial; MMN, masseter motoneurons; MoV, trigeminal motor nucleus; P, postnatal; PrV, principal sensory trigeminal nucleus; PSC, postsynaptic current; SupV, supratrigeminal region n Corresponding author. E-mail address: [email protected] (S. Nakamura).
premotor neurons that transmit such motor commands to the trigeminal motoneurons play an important role in controlling masticatory muscle activity [4,5]. The supratrigeminal (SupV) and intertrigeminal (IntV) regions, the dorsal regions of the principal sensory trigeminal nucleus (PrV), and the reticular formation dorsal to the PrV (dRt) that surround the trigeminal motor nucleus (MoV) contain premotor neurons that innervate the motoneurons of the muscles that open and close the jaw [6,7]. Furthermore, the premotor neurons in these regions that target the MoV receive afferent inputs from a number of regions, such as the orofacial structures [8–10], central pattern generator for mastication [4,11], cerebral cortex [12], amygdala [13], and lateral hypothalamus [14]. Therefore, these premotor neurons might transmit the motor commands for suckling and/or chewing. We previously reported that, in postnatal day (P) 1–5 neonatal rats, both single masseter motoneurons (MMNs) and digastric motoneurons (DMNs) receive convergent glutamatergic inputs from the SupV, IntV, PrV, and dRt,
http://dx.doi.org/10.1016/j.job.2017.01.003 1349-0079/& 2017 Japanese Association for Oral Biology. Published by Elsevier B.V. All rights reserved.
Please cite this article as: Matsuda K, et al. Premotoneuronal inputs to early developing trigeminal motoneurons. J Oral Biosci (2017), http://dx.doi.org/10.1016/j.job.2017.01.003i
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and that the lateral SupV sends burst inputs predominantly to the MMNs [15]. However, it is uncertain whether single motoneurons in juvenile (P9–12) rats receive convergent inputs from premotor neurons in the SupV, IntV, PrV, and dRt. Furthermore, it is unclear whether the premotor neurons in the lateral SupV of P9–12 rats send burst inputs predominantly to the MMNs. In this study, we investigated the synaptic inputs to MMNs and DMNs from the SupV, IntV, PrV, and dRt in P9–12 rats using the laser photolysis of caged glutamate. This allowed for the systematic stimulation of multiple confined regions, and simultaneous whole-cell recordings of motoneurons that were identified by a fluorescent dye.
2. Materials and methods 2.1. Slice preparation The experiments were performed on coronal slices of the brainstem from P9–12 Wistar rats (n ¼ 59). The slices were prepared as described in the Supplementary materials. 2.2. Retrograde labeling of jaw-closing and jaw-opening motoneurons In order to discriminate between the motoneurons innervating the muscles that open or close the jaw, we used the fluorescence labeling technique [16,17] as described in the Supplementary materials. 2.3. Patch-clamp recordings Whole-cell patch-clamp recordings were performed in the SupV neurons and the jaw-closing and jaw-opening motoneurons according to the procedures described in the Supplementary materials. 2.4. Photostimulation We systematically stimulated various sites in the slices while simultaneously performing whole-cell recordings in the SupV neurons or motoneurons. Caged glutamate was subjected to laser photolysis by a 365-nm nitrogen-pulsed Micropoint laser system equipped with galvanometer-based steering lenses (Photonic Instruments, Inc., St. Charles, IL) [cf. 15]. The laser beam was positioned by steering the lenses with the MetaMorph software (Molecular Devices, LLC, Sunnyvale, CA). After using a 40X water immersion objective to establish the whole-cell recording configuration in an MMN, DMN, or SupV neuron, we changed the 40 objective to an Olympus 4 (0.28 NA) objective. Furthermore, 4-methoxy-7-nitroindolinyl-caged L-glutamate (MNI glutamate; Tocris Bioscience, Bristol, UK) was added to the recirculating artificial cerebrospinal fluid (ACSF) to a final concentration of 300 mM in 25 mL of ACSF. In some experiments, the final MNI glutamate concentration was increased to 600 mM. All the photostimulation experiments began at least 10 min after the addition of MNI glutamate. The laser beam was focused onto an area of approximately 10 mm in diameter on the brainstem slice using the 4 objective. Single laser-beam pulses (2–6 ns) were delivered to the center of each site to trigger the focal photolysis of MNI glutamate. The photostimulation strength was always set at 70 (a.u.) in the MetaMorph software, which was the same setting used to stimulate the P1–5 rats in our previous experiment [15]. Each site was stimulated 4–8 times. The response pattern (burst PSC, low-frequency PSC, or no response) shown by over 60% of the responses across 4–8 trials was considered the representative response pattern of each sample at each stimulation site. Responses of more
than 25 pA were considered as effectively evoking postsynaptic responses. To determine the spatial resolution of the photostimulation, a single-pulsed laser beam was delivered at 5-s intervals to 100 different sites that surrounded the recording sites of the SupV neurons. The 100 sites were arranged in a 10 10 array with 40mm spaces between adjacent rows and columns (Supplementary Fig. 1A). As the brainstem is larger in P9–12 animals than in P1–5 animals, and the laser beam could only be positioned within an area of about 780 1200 mm2 in our system, we could not stimulate all the areas in the SupV, IntV, and PrV of the P9–12 animals in singlemotoneuron recording sessions. Thus, we stimulated 76 different sites in 36 slices at 2-s intervals. The photostimulation sites adjacent to the MoV, covering the SupV, IntV, and the medial border of the PrV, were arranged in an L-shape, with distances of 120 mm and 78 mm between the neighboring sites in the dorsal-ventral and medial-lateral orientations. The boundary of the MoV was identified using the 40 objective. We assigned the areas of the m-SupV, l-SupV, IntV, and PrV based on histological observations made from Nissl-stained slices. In 9 different slices, photostimulation was delivered to 40 sites in the more lateral regions, including the medial PrV. These 40 sites were arranged in an I-shape with distances of 120 mm and 78 mm between the neighboring sites in the dorsal-ventral and medial-lateral orientations (Fig. 4A). 2.5. Histological procedure The histological identification of the photostimulation sites was performed as described in the Supplementary materials. 2.6. Statistics The data are presented as the mean 7 SEM, except when specifically indicated. The data were compared within and between groups using the nonparametric Mann-Whitney U-test. Results with probability (p) values less than 0.05 were considered statistically significant. When the comparisons were performed among 4 groups, results with p values less than 0.0083 (0.05/6) were considered statistically significant. The statistical analyses were performed using the SPSS Statistics 17.0 (SPSS Japan, Tokyo, Japan) software.
3. Results 3.1. Spatial resolution of the photostimulation in P9–12 rats In order to quantify the spatial resolution of the action potentials of putative premotor neurons by a single laser-beam pulse at P9–12 rats, we examined the effects of a single stimulation pulse on the action potentials (APs) of individual putative SupV premotor neurons with a method similar to that used to examine P1– 5 rats [15]. Photostimulation applied close to the soma or proximal dendrites evoked APs (Supplementary Fig. 1A, 1D). Stimulation at some sites evoked bursts of APs at frequencies greater than 50 Hz (Supplementary Fig. 1Dc, e, 1Ec, e). For the 19 SupV neuron recordings, sites in which stimulation evoked at least one AP were located within a 103 7 43.4 mm (mean 7 SD, median: 89.4 mm; range: 40–179 mm) radius from the tip of the patch pipette, which corresponded to the location of the soma (Supplementary Fig. 1De). This value was similar to the 126 7 22.7-mm radius for P1–5 rats [15]. Thus, if the two effective photostimulation sites that evoked postsynaptic responses in a motoneuron were separated by more than twice the radius, the
Please cite this article as: Matsuda K, et al. Premotoneuronal inputs to early developing trigeminal motoneurons. J Oral Biosci (2017), http://dx.doi.org/10.1016/j.job.2017.01.003i
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Fig. 1. Mapping of the synaptic connections between premotor neurons in the supratrigeminal region (SupV) and intertrigeminal region (IntV) and a masseter motoneuron (MMN) investigated with the laser photolysis of MNI glutamate (A) Low-magnification video image of a coronal slice preparation from a postnatal-day (P) 10 rat obtained during recording overlaid by the 76 stimulation sites arranged in an L shape (upper panel) and a photomicrograph of the corresponding Nissl-stained slice (lower panel). The soma of the recorded neuron is indicated by an asterisk. Scale bar ¼ 500 mm. (B) High-magnification translucent (upper panel) and epifluorescent (lower panel) photomicrographs of the recorded MMN (indicated by arrowheads). Scale bar ¼ 50 mm. (C) Current responses corresponding to the stimulation grid shown in (A). The a, b, c, d, and e labels denote the responses that were evoked by the stimulation of the positions labeled with the same letters in (A). (D) Expanded traces of the a, b, c, d, and e response recordings shown in (C). The latencies of the currents in the a, b, c, d and e were 7.7, 9.8, 39.3, 18.9, and 0.54 ms, respectively. The slowly rising inward current in ‘e’ was evoked at a much shorter latency by stimulation of sites near the trigeminal motor nucleus (MoV), such as the ‘e’ recording shown in (C) and (D). This response was most likely evoked by the direct stimulation of the recorded MMN by uncaged glutamate [16]. This MMN exhibited the postsynaptic currents (PSCs) in response to the photostimulation of 3 different regions (l-SupV, m-SupV, and IntV). MoV, trigeminal motor nucleus; l-SupV and m-SupV, lateral and medial supratrigeminal regions, respectively; IntV, intertrigeminal region; PrV, principal sensory trigeminal nucleus; dRt, reticular formation dorsal to the PrV; Lat, lateral; Dor, dorsal.
two effective sites were considered representations of two different premotor neurons that innervated the recorded motoneuron. To avoid overestimation of the number of regions that send inputs to single motoneurons, we adopted a 252-mm radius criterion (i.e., two times 126 mm) rather than a 206-mm radius (i.e., two times 103 mm) in the evaluations of the number of inputs. 3.2. Postsynaptic responses in P9–12 motoneurons As photostimulation of the SupV and IntV evoked postsynaptic currents (PSCs) in the MMNs and DMNs in P1–5 rats [15], we examined the effects of photostimulation of the SupV and IntV on 27 MMNs and 20 DMNs in P9–12 rats. Rapidly rising inward PSCs were evoked at latencies of 5.4–40.0 ms in MMNs and DMNs when the lateral SupV (l-SupV), medial SupV (m-SupV), and IntV were stimulated (Figs. 1A–D and 2A–D). Slowly rising inward currents that were evoked at much shorter latencies of approximately 0.5 ms (below 1 ms) by stimulation applied to the sites (indicated by “e” in Fig. 1A and “d” in Fig. 2A) most likely result from the direct stimulation of the recorded motoneurons by uncaged glutamate (Fig. 1De, 2Dd) [16]. In contrast to results from P1–5 animals, lowfrequency PSCs (single, double, or multiple current responses at frequencies below 50 Hz; Fig. 1Cc, d, 1Dc, d) were the predominant responses to stimulation in the MMNs. Burst PSCs, which were defined as multiple currents comprising more than 3 sharp inward currents at high frequencies (450 Hz; Fig. 1Ca, b, 1Da, b), were less frequent. We evaluated the input profiles to single motoneurons from the SupV and IntV. Since we have previously demonstrated that the differences in electrophysiological and histological properties between the l-SupV and m-SupV [15,18], we distinguished the SupV into l-SupV and m-SupV for the evaluation
of input profiles. The upper panel of Fig. 3 shows the mean induction profiles of the burst and low-frequency PSCs that were evoked in the 27 MMNs. Photostimulation of 12 and 15 spots that approximately correspond to the l-SupV and m-SupV (outlined by dotted and dashed squares, respectively, in Fig. 3) evoked more low-frequency PSCs than burst PSCs in the MMNs (l-SupV: U ¼ 144.0, p o 0.0016; m-SupV: U ¼ 72.5, p ¼ 0.0016). Similar to the MMNs, low-frequency PSCs were evoked more often than burst PSCs (l-SupV: U ¼ 37.0, p o 0.0016; m-SupV: U ¼ 61.5, p o 0.0016; Figs. 2C, 2D, 3A) in the DMNs by l-SupV and m-SupV stimulation. However, the incidence of low-frequency PSCs in response to m-SupV stimulation was significantly higher in the MMNs compared to that in the DMNs (U ¼ 138.5, p o 0.0083; Fig. 3A, lower panel). There was no significant difference in the incidence of low-frequency PSCs in response to l-SupV stimulation between the MMNs and DMNs (U ¼ 197.5, p ¼ 0.114). Although burst PSCs were less frequent, they were evoked more in the MMNs compared with the DMNs by l-SupV and m-SupV stimulation (l-SupV: U ¼ 136.0, p ¼ 0.0016; m-SupV: U ¼ 152.5, p o 0.0083; Fig. 3A). The induction rates of either burst or lowfrequency PSCs in response to l-SupV and m-SupV stimulation were significantly higher in the MMNs compared with the DMNs (l-SupV: U ¼ 131.0, p o 0.01; m-SupV: U ¼ 116.5, p o 0.01; Fig. 3B). The profiles of the inputs to the motoneurons in P9–12 rats were similar to those in P1–5 rats. For many MMNs and DMNs, PSCs were evoked in multiple sites that were separated by more than 252 mm (Fig. 1Ca, b, c; Fig. 2Ca, b, c). We found that most of the MMNs (89%, 24/27) and DMNs (75%, 15/20) exhibited PSCs in response to photostimulation of multiple regions (Supplementary table 1).
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Fig. 2. Mapping of the synaptic connectivity between the premotor neurons and a digastric motoneuron (DMN) (A) Low-magnification video image of a coronal slice preparation from a P10 rat obtained during recording overlaid by the 76 stimulation sites arranged in an L shape (upper panel) and a photomicrograph of the corresponding Nissl-stained slice (lower panel). The soma of the recorded neuron is indicated by an asterisk. Scale bar ¼ 500 mm. (B) High-magnification translucent (upper panel) and epifluorescent (lower panel) photomicrographs of the recorded DMN (indicated by arrowheads). Scale bar ¼ 50 mm. (C) Current responses corresponding to the stimulation grid shown in (A). The a, b, c, and d labels denote the postsynaptic currents evoked by the stimulation of the positions labeled with the same letters in (A). (D) Expanded traces of the a, b, c, and d response recordings shown in (C). The latencies of the currents in the a, b, c, and d were 7.2, 12.9, 17.3, and 0.53 ms, respectively. This DMN showed PSCs in response to the photostimulation of 3 different regions (l-SupV, m-SupV, and IntV). MoV, trigeminal motor nucleus; l-SupV and m-SupV, lateral and medial supratrigeminal regions, respectively; IntV, intertrigeminal region; PrV, principal sensory trigeminal nucleus; dRt, reticular formation dorsal to the PrV; Lat, lateral; Dor, dorsal.
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Fig. 3. Induction profile of the burst or low-frequency postsynaptic currents (PSCs) that were evoked in masseter motoneurons (MMNs) and digastric motoneurons (DMNs) by photostimulation of the SupV and IntV in P9–12 rats (A) Pseudocolor maps of the incidence of burst or low-frequency PSCs in the MMNs (upper panel) and DMNs (lower panel) for each site. *p o 0.0083; **p o 0.0016. (B) Pseudocolor maps of the incidence of either burst or low-frequency PSCs in the MMNs (upper panel) and DMNs (lower panel) for each site. ‡p o 0.01. The dotted and dashed squares in (A) and (B) outline the l-SupV and m-SupV, respectively. l-SupV and m-SupV, lateral and medial supratrigeminal regions, respectively; IntV, intertrigeminal region; Lat, lateral; Dor, dorsal.
In another set of slices, photostimulation was applied to the PrV, and the responses of 9 MMNs and 8 DMNs were examined. Photostimulation of the dorsal PrV evoked low-frequency PSCs in 6 MMNs (66%, Figs. 4C, 4D) and 4 DMNs (50%, Figs. 5C, 5D), whereas burst PSCs were evoked in only 2 MMNs (22%, Fig. 4Ca, 4 Da) and 1 DMN (13%, data not shown). For both MMNs and DMNs, the incidence of burst PSCs evoked by PrV stimulation did
not differ significantly from that of low-frequency PSCs (Fig. 6). When the PrV was divided into the ventral and dorsal parts for comparison, no significant differences in the induction rate were detected between the MMNs and DMNs and/or between the burst and low-frequency PSCs. (Fig. 6). To determine the existence of inhibitory components in the photostimulation-evoked PSCs of the P9–12 motoneurons, we
Please cite this article as: Matsuda K, et al. Premotoneuronal inputs to early developing trigeminal motoneurons. J Oral Biosci (2017), http://dx.doi.org/10.1016/j.job.2017.01.003i
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5
a
a b
b MoV
PrV
a b
a
MoV
b Dor
PrV Lat
50 pA 10 ms
50 pA 200 ms
Fig. 4. Mapping of the synaptic connectivity between premotor neurons in the PrV and a masseter motoneuron (MMN) (A) Low-magnification video image of a coronal slice preparation from a P11 rat obtained during recording overlaid by the 40 stimulation sites arranged in an I shape (upper panel) and a photomicrograph of the corresponding Nissl-stained slice (lower panel). The soma of the recorded neuron is indicated by an asterisk. Scale bar ¼ 500 mm. (B) High-magnification translucent (upper panel) and epifluorescent (lower panel) photomicrographs of the recorded MMN (indicated by arrowheads). Scale bar ¼ 50 mm. (C) Current responses corresponding to the stimulation grid shown in (A). The a and b labels denote the postsynaptic currents that were evoked by stimulation of the positions labeled by the same letters in (A). (D) Expanded traces of the a and b response recordings shown in (C). The latencies of the currents in a and b were 5.7 and 21.2 ms, respectively. MoV, trigeminal motor nucleus; PrV, principal sensory trigeminal nucleus; Lat, lateral; Dor, dorsal.
recorded the PSCs evoked by photostimulation of the SupV and IntV at a holding potential of 20 mV in P9–12 MMNs (n ¼ 7). No outward PSCs were evoked by photostimulation in any of the 7 MMNs tested in P9–12 rats. 3.3. High-intensity photostimulation The low incidence of burst PSCs in P9–12 rats might have been due to decreased excitability in the premotor neurons that generate burst PSCs in trigeminal motoneurons during postnatal development. Therefore, we examined the effects of higher concentrations of MNI glutamate on PSCs in MMNs (Fig. 7A–E). Photostimulation of the l-SupV with the application of 600-mM MNI glutamate evoked burst PSCs in all 6 tested MMNs, while photostimulation at the same sites with the application of 300-mM MNI glutamate evoked low-frequency PSCs in the same MMNs (Fig. 7Ca, b, 7Da, b, 7Ea, b).
4. Discussion In the present study, we showed that low-frequency PSCs were the predominant responses in both MMNs and DMNs to photostimulation of the l-SupV and m-SupV in P9–12 rats. We have recently shown that more than one-third of the SupV premotor neurons in P1–6 rats fire at rates over 50 Hz [18]. Similarly, we have shown in both P1–5 [15] and P9–12 (present study) rats that photostimulation of the cell bodies of SupV neurons increases the firing rates of some neurons to over 50 Hz. Therefore, some
premotor neurons in the SupV probably evoke burst PSCs in trigeminal motoneurons. Furthermore, when we increased the MNI glutamate concentrations from 300 mM to 600 mM in P9–12 rats, the PSCs evoked in the MMNs by l-SupV photostimulation changed from a low-frequency pattern to a burst pattern. If the premotor neurons that innervate the trigeminal motoneurons increase in size during development like trigeminal motoneurons do [19], then the input resistances of the premotor neurons probably decrease. Thus, these results suggest that, in P9–12 rats, the uncaged glutamate induces only small depolarizations and low-frequency firing in the majority of premotor neurons in the SupV and PrV, whereas the same amount of uncaged glutamate induces large depolarizations and bursting firing patterns in many premotor neurons in the SupV and PrV in P1–5 rats. These changes in the input–output gains of the premotor neurons during photostimulation are, at least in part, responsible for the low incidence of burst PSCs in the MMNs of P9–12 rats. However, we cannot rule out the possibility that some of the premotor neurons that generate burst spikes and burst PSCs in the motoneurons of neonates may change their firing patterns during postnatal development to single spikes. We did not determine whether the evoked PSCs in the MMNs and DMNs of P9–12 rats were excitatory or inhibitory when the MNI glutamate was uncaged. Although electrical stimulation of the SupV in P1–12 rats evokes glycinergic and GABAergic PSCs in MMNs and DMNs, as well as more potent glutamatergic PSCs [16], the application of GABAA and glycine receptor antagonists in P1–5 rats did not substantially alter the induction profiles of evoked PSCs in MMNs and DMNs by photostimulation with MNI
Please cite this article as: Matsuda K, et al. Premotoneuronal inputs to early developing trigeminal motoneurons. J Oral Biosci (2017), http://dx.doi.org/10.1016/j.job.2017.01.003i
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6
a a
PrV MoV
b
b
a a PrV
b
MoV
b
Dor
50 pA Lat
10 ms 50 pA 200 ms
Fig. 5. Mapping of the synaptic connectivity between premotor neurons in the PrV and a digastric motoneuron (DMN) (A) Low-magnification video image of a coronal slice preparation from a P11 rat obtained during recording overlaid by the 40 stimulation sites arranged in an I shape (upper panel) and a photomicrograph of the corresponding Nissl-stained slice (lower panel). The soma of the recorded neuron is indicated by an asterisk. Scale bar ¼ 500 mm. (B) High-magnification translucent (upper panel) and epifluorescent (lower panel) images of the recorded DMN (indicated by arrowheads). Scale bar ¼ 50 mm. (C) Current responses corresponding to the stimulation grid shown in (A). The a and b labels denote the postsynaptic currents that were evoked by the stimulation of the positions labeled by the same letters in (A). (D) Expanded traces of a and b response recordings shown in (C). The latencies of the currents in a and b were 15.5 and 17.1 ms, respectively. MoV, trigeminal motor nucleus; PrV, principal sensory trigeminal nucleus; Lat, lateral; Dor, dorsal.
Burst PSC
Low-freq PSC
Burst+low-freq PSC
MMN n=9
Response incidence 62%
31
0
Dor
DMN n=8 Lat 200 µm Fig. 6. Induction profile of the burst or low-frequency postsynaptic currents were evoked in the masseter motoneurons (MMNs) and digastric motoneurons (DMNs) by photostimulation of the PrV in P9–12 rats (A, B) Pseudocolor maps of the incidence of burst PSCs [left panel in (A)], low-frequency [right panel in (A)], and both (B) PSCs in the MMNs (upper panel) and DMNs (lower panel) for each site. PrV, principal sensory trigeminal nucleus; Lat, lateral; Dor, dorsal.
glutamate [15]. Furthermore, we failed to evoke outward PSCs in any of the 7 MMNs tested in P9–12 rats by the uncaging of MNI glutamate (holding potential of 20 mV, patch-pipette internal solution with 0.4 mM Cl ). However, Fino et al. [20] have reported that MNI glutamate strongly blocks GABAergic responses. Therefore, in the present study, the MNI glutamate might have blocked glycinergic and GABAergic transmission to the MMNs and DMNs, and photostimulation might have evoked excitatory PSCs. In addition, we demonstrated that the photostimulation of either l-SupV or m-SupV significantly evoked PSCs in MMNs at higher rates than in DMNs in the P9–12 rats that were about to initiate chewing behavior. The activities of the jaw-closing muscles are more easily facilitated by the physical properties of food compared to the activities of the jaw-opening muscles through the activation of periodontal and spindle afferents [2,3,21]. The SupV receives periodontal and spindle afferent inputs [22–25], and the premotor neurons that target the MoV and that are rhythmically active during the jaw-closing phase of cortically induced fictive mastication have been identified in the SupV of anesthetized and immobilized rats [9]. Thus, we identified that the pattern of inputs from the l-SupV and m-SupV to the MMNs was dominant compared to the inputs to the DMNs. These results suggested that the premotor neurons in the SupV are responsible for increasing the activity of the jaw-closing muscles in response to periodontal and spindle afferent inputs during mastication. In addition, in contrast to P1–5, low-frequency PSCs in response to SupV photostimulation were predominant in the MMNs and DMNs in P9–12 rats, owing to, at least in part, developmental changes in the input-output gains of the premotor neurons. These developmental changes in
Please cite this article as: Matsuda K, et al. Premotoneuronal inputs to early developing trigeminal motoneurons. J Oral Biosci (2017), http://dx.doi.org/10.1016/j.job.2017.01.003i
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7
300 µM MNI glutamate
a
*
a
b
MoV Dor
b Med
100 pA 100 ms
600 µM MNI glutamate
a 300 µM 600 µM
a
b 300 µM
b
600 µM 50 pA 10 ms
100 pA 100 ms
Fig. 7. The effects of photostimulation of the SupV by a high concentration of MNI glutamate on the postsynaptic currents (PSCs) evoked in the MMNs (A) Low-magnification video image of a coronal slice preparation from a P12 rat obtained during recording overlaid by the 40 stimulation sites arranged in an I shape. The soma of the recorded neuron is indicated by an asterisk. Scale bar ¼ 500 mm. (B) High-magnification translucent (upper panel) and epifluorescent (lower panel) photomicrographs of the recorded MMN (indicated by arrowheads). Scale bar ¼ 50 mm. (C, D) Current responses corresponding to the stimulation grid shown in (A) recorded during the application of 300 mM MNI glutamate (C) or 600 mM MNI glutamate (D). The a and b labels denote the PSCs evoked by the stimulation of positions labeled by the same letters in (A). (E) Expanded traces of a and b response recordings shown in (C) and (D) in response to the application of 300 mM MNI glutamate (upper panel) and 600 mM MNI glutamate (lower panel), respectively. Robust PSCs were evoked in this MMN when the l-SupV was stimulated by 600 mM MNI glutamate, while photostimulation of the same area by 300 mM MNI glutamate evoked low-frequency PSCs. The latencies of the currents at the application of 300 mM and 600 mM in a were 9.2 and 5.8 ms, respectively. The latencies of the currents at the application of 300 mM and 600 mM in b were 18.3 and 20.6 ms, respectively. MoV, trigeminal motor nucleus; SupV, supratrigeminal regions; Lat, lateral; Dor, dorsal.
the properties of synaptic inputs from the SupV to trigeminal motoneurons may contribute to the proper development of chewing.
5. Conclusions We demonstrated that both P9–12 rat MMNs and DMNs received convergent inputs from the SupV, IntV, and PrV, similar to P1–5 rats, and the input–output gains of some of the premotor neurons were likely to be lower in P9–12 rats. Furthermore, we found that the MMNs received a larger number of inputs from the SupV than the DMNs did in P9–12 rats. These changes in the properties of synaptic inputs from the premotor neurons to trigeminal motoneurons may contribute to the proper development of feeding behavior.
Conflicts of interest The authors have no conflicts of interest to declare.
Ethical approval All of the experiments were approved by the International Animal Research Committee of the Showa University in accordance with the Japanese Government Law No. 105 for the care and use of laboratory animals.
Acknowledgements This work was supported by the MEXT-Supported Program for the Strategic Research Foundation at Private Universities 2012– 2016 and 2014–2018, and JSPS KAKENHI Grants JP26293397, JP15K15687, and JP16K11488.
Appendix A. Supporting information Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.job.2017.01.003.
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