Shiro Nakamura

Involvement of forebrain in parabrachial neuronal activation induced by aversively conditioned taste stimuli in the rat.

We previously have shown that forebrain inputs increase responses of amiloride-sensitive NaCl-best neurons to the conditioned stimulus (CS) in the rat parabrachial nucleus (PBN) after the establishment of conditioned taste aversion (CTA) to NaCl. In the present study, we examined the effects of aversively-conditioned NaCl taste stimulation on Fos-like immunoreactivity (FLI) in the PBN using awake intact and decerebrate rats. In Experiment 1, the CTA-trained and sham-conditioned control rats were intraorally infused with 0.1 M NaCl or 0.1 M NaCl mixed with 10(-4) M amiloride, a sodium-channel blocker. Significantly more NaCl-stimulated FLI was observed in the central medial (cms) and external lateral subnuclei (els) of PBN in the CTA-trained group than in the control group. In both groups, amiloride markedly reduced NaCl-stimulated FLI in the cms but not in the els. In Experiment 2, we found that after decerebration, there was no significant difference in FLI between the CTA-trained and sham-conditioned groups. These results suggest that (1) amirolide-sensitive taste information of NaCl projects mainly to the cms; (2) sensory information of aversive taste stimuli is likely to be represented in the els; and (3) forebrain inputs are required for elevated FLI in the PBN after CTA.

Convergent Pre-motoneuronal Inputs to Single Trigeminal Motoneurons

Because pre-motor neurons targeting trigeminal motoneurons are located in various regions, including the supratrigeminal (SupV) and intertrigeminal (IntV) regions, the principal sensory trigeminal nucleus (PrV), and the region dorsal to the PrV (dRt), a single trigeminal motoneuron may receive differential convergent inputs from these regions. We thus examined the properties of synaptic inputs from these regions to masseter motoneurons (MMNs) and digastric motoneurons (DMNs) in brainstem slice preparations obtained from P1-5 neonatal rats, using whole-cell recordings and laser photolysis of caged glutamate. Photostimulation of multiple regions within the SupV, IntV, PrV, and dRt induced post-synaptic currents (PSCs) in 14 of 19 MMNs and 18 of 26 DMNs. Furthermore, the stimulation of the lateral SupV significantly induced burst PSCs in MMNs more often than low-frequency PSCs in MMNs or burst PSCs in DMNs. Similar results were obtained in the presence of the GABAA receptor antagonist SR95531 and the glycine receptor antagonist strychnine. These results suggest that both neonatal MMNs and DMNs receive convergent glutamatergic inputs from the SupV, IntV, PrV, and dRt, and that the lateral SupV sends burst inputs predominantly to the MMNs. Such convergent pre-motoneuronal inputs to trigeminal motoneurons may contribute to the proper execution of neonatal oro-motor functions.

Properties of synaptic transmission from the reticular formation dorsal to the facial nucleus to trigeminal motoneurons during early postnatal development in rats

We previously reported that electrical stimulation of the reticular formation dorsal to the facial nucleus (RdVII) elicited excitatory masseter responses at short latencies and that RdVII neurons were antidromically activated by stimulation of the trigeminal motor nucleus (MoV), suggesting that excitatory premotor neurons targeting the MoV are likely located in the RdVII. We thus examined the properties of synaptic transmission from the RdVII to jaw-closing and jaw-opening motoneurons in horizontal brainstem preparations from developing rats using voltage-sensitive dye, patch-clamp recordings and laser photostimulation. Electrical stimulation of the RdVII evoked optical responses in the MoV. Combined bath application of the non-N-methyl-d-aspartate (non-NMDA) receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), and the NMDA receptor antagonist DL-2-amino-5-phosphonopentanoic acid (APV) reduced these optical responses, and addition of the glycine receptor antagonist strychnine and the GABA(A) receptor antagonist bicuculline further reduced the remaining responses. Electrical stimulation of the RdVII evoked postsynaptic currents (PSCs) in all 19 masseter motoneurons tested in postnatal day (P)1-4 rats, and application of CNQX and the NMDA receptor antagonist (+/-)-3(2-carboxypiperazin-4-yl)propyl-1-phosphonic acid (CPP) reduced the PSC amplitudes by more than 50%. In the presence of CNQX and CPP, the GABA(A) receptor antagonist SR95531 further reduced PSC amplitude, and addition of strychnine abolished the remaining PSCs. Photostimulation of the RdVII with caged glutamate also evoked PSCs in masseter motoneurons of P3-4 rats. In P8-11 rats, electrical stimulation of the RdVII also evoked PSCs in all 14 masseter motoneurons tested, and the effects of the antagonists on the PSCs were similar to those in P1-4 rats. On the other hand, RdVII stimulation evoked PSCs in only three of 16 digastric motoneurons tested. These results suggest that both neonatal and juvenile jaw-closing motoneurons receive strong synaptic inputs from the RdVII through activation of glutamate, glycine and GABA(A) receptors, whereas inputs from the RdVII to jaw-opening motoneurons seem to be weak.

Continuous monitoring of caspase-3 activation induced by propofol in developing mouse brain

The neurotoxicity of anesthetics on the developing brain has drawn the attention of anesthesiologists. Several studies have shown that apoptosis is enhanced by exposure to anesthesia during brain development. Although apoptosis is a physiological developmental step occurring before the maturation of neural networks and the integration of brain function, pathological damage also involves apoptosis. Previous studies have shown that prolonged exposure to anesthetics causes apoptosis. Exactly when the apoptotic cascade starts in the brain remains uncertain. If it starts during the early stage of anesthesia, even short‐term anesthesia could harm the brain. Therefore, apoptogenesis should be continuously monitored to elucidate when the apoptotic cascade is triggered by anesthesia. Here, we describe the development of a continuous monitoring system to detect caspase‐3 activation using an in vivo model. Brain slices from postnatal days 0–4 SCAT3 transgenic mice with a heterozygous genotype (n = 20) were used for the monitoring of caspase‐3 cleavage. SCAT3 is a fusion protein of ECFP and Venus connected by a caspase‐3 cleavable peptide, DEVD. A specimen from the hippocampal CA1 sector was mounted on a confocal laser microscope and was continuously superfused with artificial cerebrospinal fluid, propofol (2,6‐diisopropylphenol, 1 μM or 10 μM), and dimethyl sulfoxide. Images were obtained every hour for five hours. A pixel analysis of the ECFP/Venus ratio images was performed using a histogram showing the number of pixels with each ratio. In the histogram of the ECFP/Venus ratio, an area with a ratio > 1 indicated the number of pixels from caspase‐3‐activated CA1 neurons. We observed a shift in the histogram toward the right over time, indicating caspase‐3 activation. This right‐ward shift dramatically changed at five hours in the propofol 1 μM and 10 μM groups and was obviously different from that in the control group. Thus, real‐time fluorescence energy transfer (FRET) imaging was capable of identifying the onset of apoptosis triggered by propofol in neonatal brain slices. This model may be a useful tool for monitoring apoptogenesis in the developing brain.

Electrophysiological and morphological properties of rat supratrigeminal premotor neurons targeting the trigeminal motor nucleus

The electrophysiological and morphological characteristics of premotor neurons in the supratrigeminal region (SupV) targeting the trigeminal motor nucleus (MoV) were examined in neonatal rat brain stem slice preparations with Ca(2+) imaging, whole cell recordings, and intracellular biocytin labeling. First, we screened SupV neurons that showed a rapid rise in intracellular free Ca(2+) concentration ([Ca(2+)]i) after single-pulse electrical stimulation of the ipsilateral MoV. Subsequent whole cell recordings were generated from the screened SupV neurons, and their antidromic responses to MoV stimulation were confirmed. We divided the antidromically activated premotor neurons into two groups according to their discharge patterns during the steady state in response to 1-s depolarizing current pulses: those firing at a frequency higher (HF neurons, n = 19) or lower (LF neurons, n = 17) than 33 Hz. In addition, HF neurons had a narrower action potential and a larger afterhyperpolarization than LF neurons. Intracellular labeling revealed that the axons of all HF neurons (6/6) and half of the LF neurons (4/9) entered the MoV from its dorsomedial aspect, whereas the axons of the remaining LF neurons (5/9) entered the MoV from its dorsolateral aspect. Furthermore, the dendrites of three HF neurons penetrated into the principal sensory trigeminal nucleus (Vp), whereas the dendrites of all LF neurons were confined within the SupV. These results suggest that the types of SupV premotor neurons targeting the MoV with different firing properties have different dendritic and axonal morphologies, and these SupV neuron classes may play unique roles in diverse oral motor behaviors, such as suckling and mastication.

Coordination of NMDA-induced rhythmic activity in the trigeminal and hypoglossal nerves of neonatal mice in vitro

Suckling is a rhythmic jaw movement that is symmetrical on the left and right side and is highly coordinated with tongue movement. Thus, we investigated the neuronal mechanisms of the left/right and jaw/tongue coordinations during N-methyl-d-aspartate (NMDA)-induced fictive suckling using isolated brainstem-spinal cord preparations obtained from neonatal mice. We observed synchronous low-frequency rhythmic activity in the left/right trigeminal motor nerves, which differed from respiration, and high-frequency rhythmic trigeminal activity, which was side-independent. The low-frequency rhythmic trigeminal activity was also synchronized with the hypoglossal nerve activity. After a complete midline separation of the preparation or a partial midline transection of the brainstem from the anterior inferior cerebellar artery to the junction of the vertebral artery, the low-frequency rhythmic trigeminal activity disappeared, whereas the high-frequency rhythmic trigeminal activity and low-frequency rhythmic hypoglossal activity still remained. These results suggest that the neuronal network that generates low-frequency rhythmic activity likely contributes to the synchronized activity of the left/right jaw muscles and to the jaw/tongue muscles, where it sends its command to the trigeminal motoneurons mainly via the commissural pathway that crosses the transected midline region. Such a neuronal network may underlie the coordinated movements of the jaw and tongue during suckling.

Dark/light transition and vigilance states modulate jaw-closing muscle activity level in mice.

Bruxism is associated with an increase in the activity of the jaw-closing muscles during sleep and wakefulness. However, the changes in jaw-closing muscle activity across states of vigilance over a 24-h period are unclear. In this study, we investigated the effects of dark/light transition and sleep/wake state on EMG activity of the masseter (jaw-closing) muscle in comparison with the activity of the upper trapezius muscle (a neck muscle) over a 24-h period in mice. The activities of the masseter and neck muscles during wakefulness were much greater than during non-REM and REM sleep. In contrast, the activities of both muscles slightly, but significantly, decreased during the transition period from dark to light. Histograms of masseter activity during wakefulness and non-REM sleep showed bimodal distributions, whereas the neck muscle showed unimodal activation in all states. These results suggest that the activities of jaw-closing and neck muscles are modulated by both sleep/wake state and dark/light transition, with the latter being to a lesser degree. Furthermore, even during non-REM sleep, jaw-closing muscles display bimodal activation, which may contribute to the occurrence of exaggerated aberrant muscle activity, such as sleep bruxism.

Involvement of histaminergic inputs in the jaw-closing reflex arc

Histamine receptors are densely expressed in the mesencephalic trigeminal nucleus (MesV) and trigeminal motor nucleus. However, little is known about the functional roles of neuronal histamine in controlling oral-motor activity. Thus, using the whole-cell recording technique in brainstem slice preparations from Wistar rats aged between postnatal days 7 and 13, we investigated the effects of histamine on the MesV neurons innervating the masseter muscle spindles and masseter motoneurons (MMNs) that form a reflex arc for the jaw-closing reflex. Bath application of histamine (100 μM) induced membrane depolarization in both MesV neurons and MMNs in the presence of tetrodotoxin, whereas histamine decreased and increased the input resistance in MesV neurons and MMNs, respectively. The effects of histamine on MesV neurons and MMNs were mimicked by an H1 receptor agonist, 2-pyridylethylamine (100 μM). The effects of an H2 receptor agonist, dimaprit (100 μM), on MesV neurons were inconsistent, whereas MMNs were depolarized without changes in the input resistance. An H3 receptor agonist, immethridine (100 μM), also depolarized both MesV neurons and MMNs without changing the input resistance. Histamine reduced the peak amplitude of postsynaptic currents (PSCs) in MMNs evoked by stimulation of the trigeminal motor nerve (5N), which was mimicked by 2-pyridylethylamine but not by dimaprit or immethridine. Moreover, 2-pyridylethylamine increased the failure rate of PSCs evoked by minimal stimulation and the paired-pulse ratio. These results suggest that histaminergic inputs to MesV neurons through H1 receptors are involved in the suppression of the jaw-closing reflex although histamine depolarizes MesV neurons and/or MMNs.

Coordinated control of the tongue during suckling-like activity and respiration

The tongue can move freely and is important in oral motor functions. Tongue movement must be coordinated with movement of the hyoid, mandible, and pharyngeal wall, to which it is attached. Our previous study using isolated brainstem-spinal cord preparations showed that application of N-methyl-D-aspartate induces rhythmic activity in the hypoglossal nerve that is coincident with rhythmic activity in the ipsilateral trigeminal motor nerve. Partial or complete midline transection of the preparation only abolishes activity in the trigeminal motor nerve; therefore, the neuronal network contributing to coordinated activity of the jaw/tongue muscles is located on both sides of the preparation and sends motor commands to contralateral trigeminal motoneurons. Arterially perfused decerebrate rat preparations exhibit stable inspiratory activity in the phrenic nerve, with efferent nerves innervating the upper airway muscles (the hypoglossal nerve, a branch of the cervical spinal nerve, the external branch of the superior laryngeal nerve, and the recurrent laryngeal nerve) under normocapnic conditions (5% CO2). During hypercapnia (8% CO2), pre-inspiratory discharges appear in all nerves innervating upper airway muscles. Such coordinated activity in the pre-inspiratory phase contributes to dilation of the upper airway and improves hypercapnia.

Generation of orthotopically functional salivary gland from embryonic stem cells

Organoids generated from pluripotent stem cells are used in the development of organ replacement regenerative therapy by recapitulating the process of organogenesis. These processes are strictly regulated by morphogen signalling and transcriptional networks. However, the precise transcription factors involved in the organogenesis of exocrine glands, including salivary glands, remain unknown. Here, we identify a specific combination of two transcription factors (Sox9 and Foxc1) responsible for the differentiation of mouse embryonic stem cell-derived oral ectoderm into the salivary gland rudiment in an organoid culture system. Following orthotopic transplantation into mice whose salivary glands had been removed, the induced salivary gland rudiment not only showed a similar morphology and gene expression profile to those of the embryonic salivary gland rudiment of normal mice but also exhibited characteristics of mature salivary glands, including saliva secretion. This study suggests that exocrine glands can be induced from pluripotent stem cells for organ replacement regenerative therapy.Functional salivary glands have not been generated from embryonic stem cells (mESCs) to date. Here the authors demonstrate directed in vitro differentiation of mESCs to oral ectoderm and salivary gland rudiments that form mature, functional salivary glands after orthotopic transplantation.