Shared GABA transmission pathology in dopamine agonist- and antagonist-induced dyskinesia.

Dyskinesia is involuntary movement caused by long-term medication with dopamine-related agents: the dopamine agonist 3,4-dihydroxy-L-phenylalanine (L-DOPA) to treat Parkinson's disease (L-DOPA-induced dyskinesia [LID]) or dopamine antagonists to treat schizophrenia (tardive dyskinesia [TD]). However, it remains unknown why distinct types of medications for distinct neuropsychiatric disorders induce similar involuntary movements. Here, we search for a shared structural footprint using magnetic resonance imaging-based macroscopic screening and super-resolution microscopy-based microscopic identification. We identify the enlarged axon terminals of striatal medium spiny neurons in LID and TD model mice. Striatal overexpression of the vesicular gamma-aminobutyric acid transporter (VGAT) is necessary and sufficient for modeling these structural changes; VGAT levels gate the functional and behavioral alterations in dyskinesia models. Our findings indicate that lowered type 2 dopamine receptor signaling with repetitive dopamine fluctuations is a common cause of VGAT overexpression and late-onset dyskinesia formation and that reducing dopamine fluctuation rescues dyskinesia pathology via VGAT downregulation.

Optical imaging of neurons related to fictive swallowing using GCaMP6f in an arterially perfused rat preparation.

OBJECTIVE: It is difficult to comprehensively study the activity patterns and distribution of neurons in the brainstem that control the act of swallowing, as they are located deep in the brain. In this study, we aimed to evaluate the usefulness of calcium imaging using GCaMP6f in arterially perfused preparations to study the activity of swallowing-related neurons in the brainstem. METHODS: Arterially perfused rat preparations were prepared 3-4 weeks after the injection of a neuron-specific virus expressing GCaMP6f. Fictive swallowing was induced by repetitive electrical stimulation of the superior laryngeal nerve (SLN). Simultaneously, the activity of GCaMP6f-expressing neurons in the dorsal brainstem, between 0.1 and 4.8 mm rostral to the obex, was assessed by changes in the intracellular calcium concentration using confocal laser microscopy. RESULTS: Neurons responding to stimulation of the SLN included swallowing-related neurons (48%), which showed an increase in fluorescence intensity at the time of swallowing bursts in the cervical vagus nerve, and stimulation-related neurons (52%), which showed an increase in fluorescence intensity through stimulation, regardless of the swallowing bursts. Despite a broad search area, swallowing-related neurons were localized exclusively in and around the solitary nucleus. In contrast, most stimulation-related neurons were located in the brainstem reticular formation, which is more rostral than the solitary nucleus. CONCLUSIONS: Calcium imaging using GCaMP in arterially perfused rat preparations is useful for an efficient search of the activity pattern and distribution of neurons located in a wide area of the brainstem.

Involvement of ghrelin in the regulation of swallowing motor activity in an arterially perfused rat preparation.

Ghrelin, a peripheral peptide produced in the stomach, is involved in the neural networks that control food intake. Alterations in motor components, such as swallowing, are believed to be significant in the regulation food intake by orexigenic signals. However, there has been no detailed investigation of the relationship between ghrelin and swallowing activities induced in motor nerves innervating the pharyngeal and laryngeal muscles. In this study, we examined the effects of ghrelin administration on swallowing motor activity in arterially perfused rats. Injection of distilled water (0.5 ml) into the oral cavity or electrical stimulation of the superior laryngeal nerve evoked swallowing motor activity in the cervical vagus nerve. Administration of ghrelin (6 nM), but not des-acylated ghrelin (6 nM), into the perfusate increased the peak burst amplitude and burst duration, and shortened the first burst interval of water injection-induced swallowing. These ghrelin-induced changes in swallowing motor activity were blocked by the administration of JMV2959 (6 µM), a growth hormone secretagogue receptor antagonist. In preparations in which the hypothalamus was removed, ghrelin had no effect on swallowing motor activity. Furthermore, ghrelin-induced changes were counteracted by the administration of BIBO3304 (1 µM) or L-152,804 (1 µM), antagonists of neuropeptide Y Y1 and Y5 receptors, respectively, which are essential for ghrelin-induced enhancement of food intake. Ghrelin also increased the peak burst amplitude and burst duration of the swallowing motor activity evoked by electrical stimulation of the superior laryngeal nerve, although the effects of ghrelin on the number of swallowing bursts and burst intervals varied with stimulus intensity. These results suggest that ghrelin enhances the magnitude and frequency of bursts of swallowing motor activity by acting via the hypothalamic neural network, and that neuropeptide Y Y1 and Y5 receptors are involved in this enhancement.

Intrinsic properties and synaptic connectivity of Phox2b-expressing neurons in rat rostral parvocellular reticular formation.

The paired-like homeobox 2b gene (Phox2b) is critical for the development of the autonomic nervous system. We have previously demonstrated the distinct characteristics of Phox2b-expressing (Phox2b+) neurons in the reticular formation dorsal to the trigeminal motor nucleus (RdV), which are likely related to jaw movement regulation. In this study, we focused on Phox2b+ neurons in the rostral parvocellular reticular formation (rPCRt), a critical region for controlling orofacial functions, using 2-11-day-old Phox2b-EYFP rats. Most Phox2b+ rPCRt neurons were glutamatergic, but not GABAergic or glycinergic. Approximately 65 % of Phox2b+ rPCRt neurons fired at a low frequency, and approximately 24 % of Phox2b+ rPCRt neurons fired spontaneously, as opposed to Phox2b+ RdV neurons. Stimulation of the RdV evoked inward postsynaptic currents in more than 50 % of Phox2b+ rPCRt neurons, while only one Phox2b+ rPCRt neuron responded to stimulation of the nucleus of the solitary tract. Five of the 10 Phox2b+ neurons sent their axons that ramified within the trigeminal motor nucleus (MoV). Of these, the axons of the two neurons terminated within both the MoV and rPCRt. Our findings suggest that Phox2b+ rPCRt neurons have distinct electrophysiological and synaptic properties that may be involved in the motor control of feeding behavior.

Developmental changes in GABAergic and glycinergic synaptic transmission to rat motoneurons innervating jaw-closing and jaw-opening muscles.

Trigeminal motoneurons (MNs) innervating the jaw-closing and jaw-opening muscles receive numerous inhibitory synaptic inputs from GABAergic and glycinergic neurons, which are essential for oromotor functions, such as the orofacial reflex, suckling, and mastication. The properties of the GABAergic and glycinergic inputs of these MNs undergo developmental alterations during the period in which their feeding behavior proceeds from suckling to mastication; however, the detailed characteristics of the developmental patterns of GABAergic and glycinergic transmission in these neurons remain to be elucidated. This study was conducted to investigate developmental changes in miniature inhibitory postsynaptic currents (mIPSCs) in masseter (jaw-closing) and digastric (jaw-opening) MNs using brainstem slice preparations obtained from Wistar rats on postnatal day (P)2-5, P9-12, and P14-17. The frequency and amplitude of glycinergic mIPSCs substantially increased with age in both the masseter and digastric MNs. The rise time and decay time of glycinergic mIPSCs in both MNs decreased during development. In contrast, the frequency of GABAergic components in masseter MNs was higher at P2-5 than at P14-17, whereas that in the digastric MNs remained unchanged throughout the postnatal period. The proportion of currents mediated by GABA-glycine co-transmission was higher at P2-5, and then it decreased with age in both MNs. These results suggest that characteristics related to the development of inhibitory synaptic inputs differ between jaw-closing and jaw-opening MNs and between GABAergic and glycinergic currents. These distinct developmental characteristics may contribute to the development of feeding behaviors.

Postnatal Maturation of Glutamatergic Inputs onto Rat Jaw-closing and Jaw-opening Motoneurons.

Motoneurons that innervate the jaw-closing and jaw-opening muscles play a critical role in oro-facial behaviors, including mastication, suckling, and swallowing. These motoneurons can alter their physiological properties through the postnatal period during which feeding behavior shifts from suckling to mastication; however, the functional synaptic properties of developmental changes in these neurons remain unknown. Thus, we explored the postnatal changes in glutamatergic synaptic transmission onto the motoneurons that innervate the jaw-closing and jaw-opening musculatures during early postnatal development in rats. We measured miniature excitatory postsynaptic currents (mEPSCs) mediated by non-NMDA receptors (non-NMDA mEPSCs) and NMDA receptors in the masseter and digastric motoneurons. The amplitude, frequency, and rise time of non-NMDA mEPSCs remained unchanged among postnatal day (P)2-5, P9-12, and P14-17 age groups in masseter motoneurons, whereas the decay time dramatically decreased with age. The properties of the NMDA mEPSCs were more predominant at P2-5 masseter motoneurons, followed by reduction as neurons matured. The decay time of NMDA mEPSCs of masseter motoneurons also shortened remarkably across development. Furthermore, the proportion of NMDA/non-NMDA EPSCs induced in response to the electrical stimulation of the supratrigeminal region was quite high in P2-5 masseter motoneurons, and then decreased toward P14-17. In contrast to masseter motoneurons, digastric motoneurons showed unchanged properties in non-NMDA and NMDA EPSCs throughout postnatal development. Our results suggest that the developmental patterns of non-NMDA and NMDA receptor-mediated inputs vary among jaw-closing and jaw-opening motoneurons, possibly related to distinct roles of respective motoneurons in postnatal development of feeding behavior.

Responses evoked by electrical stimulation of the brainstem reticular formation in the jaw-opening and hypoglossal motor nerves of an arterially perfused rat preparation.

The interneuronal system in the brainstem reticular formation plays an important role in elaborate muscle coordination during various orofacial motor behaviors. In this study, we examined the distribution in the brainstem reticular formation of the sites that induce monosynaptic motor activity in the mylohyoid (jaw-opening) and hypoglossal nerves using an arterially perfused rat preparation. Electrical stimulation applied to 286 and 247 of the 309 sites in the brainstem evoked neural activity in the mylohyoid and hypoglossal nerves, respectively. The mean latency of the first component in the mylohyoid nerve response was significantly shorter than that in the hypoglossal nerve response. Moreover, the latency histogram of the first component in the hypoglossal nerve responses was bimodal, which was separated by 4.0 ms. The sites that induced short-latency (<4.0 ms) motor activity in the mylohyoid nerve and the hypoglossal nerve were frequently distributed in the rostral portion and the caudal portion of the brainstem reticular formation, respectively. Such difference in distributions of short-latency sites for mylohyoid and hypoglossal nerve responses likely corresponds to the distribution of excitatory premotor neurons targeting mylohyoid and hypoglossal motoneurons.

Serotonin1B receptor-mediated presynaptic inhibition of proprioceptive sensory inputs to jaw-closing motoneurons.

The proprioceptive sensory inputs from neurons in the mesencephalic trigeminal nucleus (MesV) to masseter motoneurons (MMNs) play an important role in regulating masseter muscle activity during mastication. Several histological studies have shown that serotonin (5-HT) fibers densely innervate both the MesV and the trigeminal motor nucleus. However, the functional roles of 5-HT in the regulation of the excitatory synaptic inputs from MesV afferents to MMNs remain to be clarified. Thus, using the whole-cell recording technique in brainstem slice preparations from juvenile Wistar rats aged between postnatal days 8 and 12, we examined the effects of 5-HT on the excitatory synaptic inputs from MesV afferents to MMNs. Bath application of 5-HT reduced the peak amplitude of excitatory postsynaptic potentials evoked in MMNs by electrical stimulation of the MesV afferents (eEPSPs), and this inhibitory effect of 5-HT on eEPSPs was replicated with the 5-HT1B receptor agonist CP-93129 but not by the 5-HT1A receptor agonist 8-OH-DPAT. Moreover, the 5-HT1B receptor antagonist SB-224289 but not the 5-HT1A receptor antagonist WAY-100635 antagonized the inhibitory effect of 5-HT on eEPSPs. CP-93129 increased the paired-pulse ratio and decreased the frequency of miniature excitatory postsynaptic currents (mEPSCs), while it did not alter the mEPSC amplitude. These results suggest that presynaptic 5-HT1B receptors are involved in the inhibition of the excitatory synaptic inputs from MesV afferents to MMNs. Such inhibition may regulate MesV afferent activity during mastication.

5-HT2A receptor activation enhances NMDA receptor-mediated glutamate responses through Src kinase in the dendrites of rat jaw-closing motoneurons.

KEY POINTS: 5-HT increases the excitability of brainstem and spinal motoneurons, including the jaw-closing motoneurons, by depolarizing the membrane potential and decreasing the medium-duration afterhyperpolarization. In this study, we focused on how 5-HT enhances postsynaptic glutamatergic responses in the dendrites of the jaw-closing motoneurons. We demonstrate that 5-HT augments glutamatergic signalling by enhancing the function of the GluN2A-containing NMDA receptor (NMDAR) through the activation of 5-HT2A receptors (5-HT2A Rs) and Src kinase. To enhance glutamatergic responses, activation of the 5-HT2A Rs must occur within ∼60 µm of the location of the glutamate responses. 5-HT inputs to the jaw-closing motoneurons can significantly vary their input-output relationship, which may contribute to wide-range regulation of contractile forces of the jaw-closing muscles. ABSTRACT: Various motor behaviours are modulated by 5-HT. Although the masseter (jaw-closing) motoneurons receive both glutamatergic and serotonergic inputs, it remains unclear how 5-HT affects the glutamatergic inputs to the motoneuronal dendrites. We examined the effects of 5-HT on postsynaptic responses evoked by single- or two-photon uncaging of caged glutamate (glutamate responses) to the dendrites of masseter motoneurons in postnatal day 2-5 rats of either sex. Application of 5-HT induced membrane depolarization and enhanced the glutamate-response amplitude. This enhancement was mimicked by the 5-HT2A receptor (5-HT2A R) agonist and was blocked by the 5-HT2A/2C R antagonist. However, neither the 5-HT2B R nor the 5-HT2C R agonists altered glutamate responses. Blockade of the NMDA receptors (NMDARs), but not AMPA receptors, abolished the 5-HT-induced enhancement. Furthermore, the selective antagonist for the GluN2A subunit abolished the 5-HT-induced enhancement. 5-HT increased GluN2A phosphorylation, while the Src kinase inhibitor reduced the 5-HT-induced enhancement and GluN2A phosphorylation. When exposure to the 5-HT2A R agonist was targeted to the dendrites, the enhancement of glutamate responses was restricted to the loci of the dendrites near the puff loci. Electron microscopic immunohistochemistry revealed that both the NMDARs and the 5-HT2A Rs were close to each other in the same dendrite. These results suggest that activation of dendritic 5-HT2A Rs enhances the function of local GluN2A-containing NMDARs through Src kinase. Such enhancement of the glutamate responses by 5-HT may contribute to wide-range regulation of contractile forces of the jaw-closing muscles.