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. Author manuscript; available in PMC: 2018 Feb 7.
Published in final edited form as: Cell Metab. 2017 Jan 5;25(2):322–334. doi: 10.1016/j.cmet.2016.12.002

Medullary Reticular Neurons Mediate Neuropeptide Y-Induced Metabolic Inhibition and Mastication

Yoshiko Nakamura 1, Yuchio Yanagawa 2, Shaun F Morrison 3, Kazuhiro Nakamura 1,4,5,*
PMCID: PMC5299028  NIHMSID: NIHMS836089  PMID: 28065829

SUMMARY

Hypothalamic neuropeptide Y (NPY) elicits hunger responses to increase the chances of surviving starvation: an inhibition of metabolism and an increase in feeding. Here we elucidate a key central circuit mechanism through which hypothalamic NPY signals drive these hunger responses. GABAergic neurons in the intermediate and parvicellular reticular nuclei (IRt/PCRt) of the medulla oblongata, which are activated by NPY-triggered neural signaling from the hypothalamus, potentially through the nucleus tractus solitarius, mediate the NPY-induced inhibition of metabolic thermogenesis in brown adipose tissue (BAT) via their innervation of BAT sympathetic premotor neurons. Intriguingly, the GABAergic IRt/PCRt neurons innervating the BAT sympathetic premotor region also innervate the masticatory motor region, and stimulation of the IRt/PCRt elicits mastication and increases feeding as well as inhibits BAT thermogenesis. These results indicate that GABAergic IRt/PCRt neurons mediate hypothalamus-derived hunger signaling by coordinating both autonomic and feeding motor systems to reduce energy expenditure and to promote feeding.

Graphical Abstract

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INTRODUCTION

Hunger stimulates appetite and reduces energy expenditure—innate physiological responses to improve energy balance between feeding bouts and to increase the chances of survival under more extreme starvation conditions. These hunger responses are driven by hypothalamic neural circuits, in which neuropeptide Y (NPY) plays a key role (Loh et al., 2015). NPY/agouti-related peptide (AgRP)-containing neurons in the arcuate nucleus of the hypothalamus are activated by peripheral hunger signaling mediated by hormones such as ghrelin secreted by the stomach (Kohno et al., 2003; Chen et al., 2015), and they release NPY from their axonal projections to other hypothalamic sites including the paraventricular hypothalamic nucleus (PVH) (Kalra et al., 1991). The hypothalamic action of NPY elicits an increase in food intake as well as a reduction in metabolism (Stanley and Leibowitz, 1985; Walker and Romsos, 1993; Atasoy et al., 2012). However, the central circuit mechanism by which the NPY-mediated hunger signaling from the hypothalamus coordinates the autonomic and somatic motor systems to reduce energy expenditure and to drive the consummatory responses remains unknown.

Hypothalamic release of NPY, including into the PVH, suppresses energy expenditure in excess of the basal metabolic rate by inhibiting active heat production, such as that occurring in brown adipose tissue (BAT) when animals, including humans (Betz and Enerbäck, 2015), are in a sub-thermoneutral environment (Egawa et al., 1991; Székely et al., 2005). BAT thermogenesis is an adaptive response to maintain body temperature in cold environments and to develop fever during infection, driven by the central sympathetic neural pathway descending from the thermoregulatory center in the preoptic area (Nakamura 2011; Morrison et al., 2014). In this pathway, BAT sympathetic premotor neurons in the rostral medullary raphe region (rMR), including the rostral raphe pallidus and raphe magnus nuclei, play a key role by integrating information from various brain regions to provide excitatory commands to the spinal output system to drive BAT thermogenesis (Nakamura et al., 2004). These premotor neurons express vesicular glutamate transporter 3 (VGLUT3) and are activated by cold exposure or central administration of prostaglandin E2 (PGE2), a pyrogenic mediator (Nakamura et al., 2004). The important role of the sympathetic premotor neurons in the control of BAT thermogenesis and energy expenditure led us to hypothesize that NPY-mediated hunger signaling from the hypothalamus inhibits the premotor neurons to reduce the thermogenic sympathetic outflow to BAT.

In the present study, we sought to elucidate the central circuit mechanism by which the hypothalamic NPY-mediated hunger signal acts on the medullary motor systems to inhibit BAT thermogenesis and to stimulate feeding in a coordinated manner. To this end, we first sought to identify the neurons that mediate NPY-triggered signaling from the hypothalamus to inhibit BAT sympathetic premotor neurons, by using in vivo electrophysiological, neuroanatomical and chemogenetic techniques. Furthermore, we examined the possibility that the identified neurons are also involved in the NPY-stimulated feeding occurring synchronously with the inhibition of BAT thermogenesis.

RESULTS

NPY-Triggered Signaling from the PVH Inhibits BAT Thermogenesis through GABAergic Inputs to Sympathetic Premotor Neurons

To test the hypothesis that NPY-mediated hunger signaling from the hypothalamus inhibits BAT thermogenesis through inhibition of the sympathetic premotor outflow from the rMR, we examined the effect of an injection of NPY into the rat hypothalamus on BAT thermogenesis evoked by stimulating sympathetic premotor neurons in the rMR. Consistent with the view that the sympathetic premotor drive for BAT thermogenesis is determined by the balance between excitatory (glutamatergic) and inhibitory (GABAergic) inputs to the premotor neurons (Nakamura 2011) (Figure 1A), either stimulation of NMDA glutamate receptors or blockade of GABAA receptors in the rMR elicited increases in BAT sympathetic nerve activity (SNA), BAT temperature, expired CO2, heart rate and mean arterial pressure (Figure 1 and Figure S1). Nanoinjection of NPY into the PVH eliminated all the thermogenic and cardiovascular responses to the glutamatergic excitation of premotor neurons in the rMR with NMDA, but had no inhibitory effect on those evoked by blocking GABAA receptors in the rMR with bicuculline (Figure 1 and Figure S1). These results indicate that an action of NPY on PVH neurons leads to an inhibition of BAT thermogenesis and cardiovascular tone through a GABAergic inhibition of sympathetic premotor neurons in the rMR.

Figure 1. Hypothalamic NPY Inhibits BAT Thermogenesis through GABAergic Transmission to Sympathetic Premotor Neurons.

Figure 1

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(A) Sympathetic premotor neurons in the rMR are controlled by glutamatergic excitatory and GABAergic inhibitory inputs.

(B–D) NPY injection into the rat PVH suppressed increases in BAT SNA induced by NMDA nanoinjections into the rMR (C), but not that induced by blockade of GABAA receptors in the rMR with a bicuculline nanoinjection (D). Arrows in (B) show representative injection sites in the PVH (top) and rMR (bottom). Scale bars, 0.3 mm. 3V, third ventricle; py, pyramidal tract; RMg, raphe magnus nucleus; rRPa, rostral raphe pallidus nucleus.

(E) NMDA-induced changes in BAT SNA before and after NPY injection into the PVH (n = 5) are expressed as the area under the curve of the ‘power/4 s’ trace (C) above the pre-injection level (subtracted AUC) for 5 min after an NMDA injection. For data analysis, see Supplemental Experimental Procedures. *P < 0.05 (unpaired t-test).

(F) Bicuculline-evoked (Bic-evoked) changes in BAT SNA and the effect of NPY injection into the PVH [from (D), n = 5]. ns: not significant (paired t-test vs Bic-evoked value). All values are means ± SEM. See also Figure S1 for all the injection sites and changes in other physiological variables.

Medullary GABAergic Neurons Innervating BAT Sympathetic Premotor Neurons

To identify the source of the GABAergic inputs to the rMR through which NPY-mediated signaling from the PVH inhibits BAT sympathetic premotor neurons, we performed neural tract tracing studies. Cholera toxin b-subunit (CTb), a retrograde tracer, was injected into the rMR of Gad1 (GAD67)-Gfp knock-in mice (Figure 2A), in which GABAergic neurons were labeled with GFP (Tamamaki et al., 2003). CTb labeling was found in many GFP-positive neurons in the intermediate reticular nucleus (IRt) and the parvicellular reticular nucleus (PCRt) of the medulla oblongata at the rostrocaudal levels around the rostral pole of the inferior olivary complex (Figure 2B and C). Cell counting at three rostrocaudal levels (Figure 2C) showed that 42 ± 2% (n = 3 mice) of CTb-labeled IRt/PCRt neurons (45 ± 9 of 108 ± 16 cells) expressed GFP. Although a few double-positive neurons were also distributed in regions surrounding the IRt/PCRt (Figure 2C), such as the rostral ventrolateral medulla (RVL) (4 ± 1 GFP-positive in 30 ± 7 CTb-labeled cells) and the nucleus tractus solitarius (NTS) (6 ± 1 GFP-positive in 19 ± 2 CTb-labeled cells), no other medullary regions contained a substantial number of double-positive neurons.

Figure 2. Putative Synaptic Inputs from GABAergic IRt/PCRt Neurons to Sympathetic Premotor Neurons in the rMR.

Figure 2

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(A–C) Retrograde tracing from the rMR in Gad1-Gfp knock-in mice. CTb injection into the rMR ([A], red) resulted in labeling of GFP-expressing neurons in the IRt/PCRt with CTb ([B], arrowheads). Drawings (C) show the distribution of CTb-labeled and GFP-expressing neurons in the medulla oblongata (representative of 3 mice). Scale bars, 30 μm (B), 0.5 mm (C). Amb, ambiguus nucleus; cRPa, caudal raphe pallidus nucleus; Gi, gigantocellular reticular nucleus; Giα, alpha part of the gigantocellular reticular nucleus; Giv, ventral part of the gigantocellular reticular nucleus; icp, inferior cerebellar peduncle; IO, inferior olivary complex; LPGi, lateral paragigantocellular nucleus; ml, medial lemniscus; mlf, medial longitudinal fasciculus; ROb, raphe obscurus nucleus; sp5, spinal trigeminal tract; Sp5, spinal trigeminal nucleus.

(D and E) Anterograde tracing from the IRt/PCRt to the rMR in rats. PHA-L was injected bilaterally into the IRt/PCRt ([D], green). Arrowheads in the pseudocolored confocal images (E) indicate apposition of PHA-L-labeled, VGAT-containing axon swellings to dendrites and cell bodies of VGLUT3-immunoreactive neurons (asterisk, cell body) in the rMR (representative of 3 rats). Scale bar, 5 μm.

Anterograde tracing from the IRt/PCRt with Phaseolus vulgaris leucoagglutinin (PHA-L) in rats (Figure 2D) labeled axon fibers and swellings in the rMR that contained vesicular GABA transporter (VGAT), a marker for GABAergic neurons, and that were closely associated with neuronal cell bodies and dendrites of VGLUT3-immunoreactive sympathetic premotor neurons (Figure 2E). Such putative synaptic connections of IRt/PCRt-derived GABAergic axons with VGLUT3-immunoreactive neurons were frequently found around a midline portion of the rMR (approximately 2–10 obvious appositions found by a brief confocal scanning of a 400 μm × 400 μm area). Because this microscopy likely missed many contacts of PHA-L-labeled fibers with the scarcely immunoreactive dendrites of VGLUT3-expressing neurons, more IRt/PCRt-derived GABAergic axons probably connect with those sympathetic premotor neurons. These retrograde and anterograde tracing results strongly support a direct innervation of BAT sympathetic premotor neurons in the rMR by GABAergic neurons in the IRt/PCRt.

Stimulation of GABAergic Neurons in the IRt/PCRt Inhibits BAT Thermogenic and Cardiac Responses

To investigate the role of neurons in the IRt/PCRt in the regulation of BAT thermogenesis, we examined the effect of stimulation of IRt/PCRt neurons on febrile BAT thermogenesis. Nanoinjection of PGE2 into the rat preoptic area elicited intense BAT thermogenesis as well as metabolic (expired CO2) and cardiovascular stimulation (Figure 3) (Nakamura et al., 2005). The PGE2-evoked increases in BAT thermogenesis, metabolism and heart rate were reversed by unilateral stimulation of IRt/PCRt neurons with a nanoinjection of bicuculline, but not with saline (Figure 3A–D). There was no significant change in arterial pressure due to stimulation of IRt/PCRt neurons (Figure 3C), although it did increase in some cases (Figure 3A; Marchenko and Sapru, 2003). Mapping with bicuculline nanoinjections (Figure 3E) revealed the narrow distribution of the relevant IRt/PCRt neurons (2.6–2.8 mm caudal to the interaural line), since a rostral or caudal shift of only 0.3–0.5 mm in the position of the nanoinjections in the IRt/PCRt resulted in a drastically reduced inhibitory effect on the evoked BAT SNA.

Figure 3. Stimulation of IRt/PCRt Neurons Reverses Febrile BAT Thermogenesis, Metabolism and Tachycardia.

Figure 3

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(A and B) Unilateral nanoinjection of bicuculline (A), but not saline (B), into the rat IRt reversed increases in BAT SNA, BAT temperature (TBAT), expired (Exp.) CO2 and heart rate (HR) evoked by PGE2 injection into the preoptic area (POA). AP, arterial pressure; Trec, rectal temperature.

(C) PGE2-evoked changes in physiological variables and the effect of bicuculline or saline injection into the IRt/PCRt at interaural −2.6 ~ −2.8 mm (n = 4 for saline, n = 5 for bicuculline). The boxed map shows the sites of injections chosen for these group data from (E). The PGE2-evoked “Increase” values and the “Effect” values after the injection into the IRt/PCRt are shown. For data analysis, see Supplemental Experimental Procedures. ns: not significant, *P < 0.05 (paired t-test vs Increase values). All values are means ± SEM.

(D) Representative views of injection sites in the POA (left) and IRt (right). Scale bars, 0.5 mm. ac, anterior commissure; MnPO, median preoptic nucleus; MPO, medial preoptic area; ox, optic chiasm.

(E) Composite drawings that map sites of saline and bicuculline injections with their inhibitory effects on PGE2-stimulated BAT SNA. 7, facial nucleus.

Similar to NPY injection into the PVH (Figure 1D), stimulation of IRt/PCRt neurons failed to inhibit the BAT thermogenic, metabolic and tachycardic responses evoked by antagonizing GABAA receptors in the rMR with bicuculline (Figure S2). In contrast, stimulation of nearby RVL neurons did inhibit the rMR bicuculline-evoked responses (Figure S2; Cao et al., 2010) as well as the PGE2-evoked BAT SNA (Figure 3E), indicating that IRt/PCRt neurons and RVL neurons are involved in distinct BAT sympathoinhibitory circuit mechanisms.

To directly examine whether activation of the GABAergic neurons in the IRt/PCRt inhibits BAT thermogenic and cardiac responses, we selectively stimulated GABAergic neurons in the IRt/PCRt in vivo by using a chemogenetic technique with hM3Dq, a Gq-coupled designer receptor exclusively activated by designer drug (DREADD; Roth 2016), and examined the effects on BAT thermogenic and cardiac responses to cooling in mice. To achieve GABAergic neuron-specific expression of hM3Dq in the IRt/PCRt, adeno-associated virus (AAV) expressing the Cre-dependent hM3Dq-mCherry transgene (AAV-EF1α-DIO-hM3Dq-mCherry) was injected into the IRt/PCRt of Gad2-IRES-Cre knock-in mice, in which Cre recombinase was targeted to the Gad2 (GAD65) gene (Taniguchi et al., 2011). Confirming the high selectivity of the expression of hM3Dq-mCherry in GABAergic neurons, injection of the AAV into the IRt/PCRt of Gad2-IRES-Cre knock-in mice crossed with the Gad1-Gfp knock-in strain resulted in 90% of hM3Dq-mCherry-expressing cells in the IRt/PCRt being GFP-positive, GABAergic neurons (Figure 4A; 169 GFP-positive in 188 hM3Dq-mCherry-expressing cells counted in 3 sections). Repeated skin cooling challenges to Gad2-IRES-Cre knock-in mice in which the AAV was bilaterally injected into the IRt/PCRt (Figure 4B) consistently increased BAT temperature and heart rate (Figure 4C). However, the cooling-induced BAT thermogenesis and tachycardia were eliminated following bilateral nanoinjections of the hM3Dq agonist, clozapine-N-oxide (CNO), into the IRt/PCRt to stimulate local GABAergic neurons, but not following vehicle injections into the IRt/PCRt (Figure 4C–E). Subsequent to the CNO treatment, inhibition of IRt/PCRt neurons with bilateral nanoinjections of muscimol, a GABAA receptor agonist widely used in vivo as a neuronal inhibitor, recovered the cooling-induced BAT thermogenesis and tachycardia (Figure 4C–E). Together with our tracing studies showing the IRt/PCRt–rMR GABAergic projection, these physiological results indicate that GABAergic neurons in the IRt/PCRt can inhibit BAT thermogenic and cardiac responses through their projections to sympathetic premotor neurons in the rMR.

Figure 4. Chemogenetic Stimulation of GABAergic IRt/PCRt Neurons Eliminates Cooling-Induced BAT Thermogenesis and Tachycardia.

Figure 4

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(A) Selective expression of hM3Dq-mCherry (arrows) in GFP-expressing GABAergic neurons in the IRt/PCRt of a Gad2-IRES-Cre/Gad1-Gfp double knock-in mouse injected with AAV-EF1α-DIO-hM3Dq-mCherry into the IRt/PCRt. Scale bar, 30 μm.

(B) Distribution of hM3Dq-mCherry-expressing neurons in a Gad2-IRES-Cre knock-in mouse injected with AAV-EF1α-DIO-hM3Dq-mCherry into the IRt/PCRt. Scale bar, 0.5 mm.

(C) Effects of bilateral nanoinjections of vehicle, CNO and muscimol into the IRt/PCRt of a Gad2-IRES-Cre knock-in mouse injected with AAV-EF1α-DIO-hM3Dq-mCherry on BAT thermogenesis and tachycardia induced by trunk skin cooling (blue periods).

(D) Sites of drug injections. Vehicle, CNO and muscimol were injected at the same sites. The left side of the symmetric bilateral injection sites is shown.

(E) Cooling-induced changes in TBAT and HR (n = 6). For data analysis, see Supplemental Experimental Procedures. *P < 0.05, **P < 0.01 (Tukey’s multiple comparisons test following repeated measures one-way ANOVA). All values are means ± SEM.

Central NPY Activates GABAergic IRt/PCRt Neurons Projecting to the rMR

Whether GABAergic IRt/PCRt neurons projecting to the rMR are involved in central NPY-triggered neural circuit mechanisms was examined by performing in vivo unit recording in rats. Using collision tests (Figure 5A), we identified 11 IRt/PCRt neurons that were antidromically activated by electrical stimulation in the rMR. Ten of these IRt/PCRt neurons projecting to the rMR increased their firing rates in response to injection of NPY into the lateral ventricle (Figure 5B; before NPY, 5.3 ± 2.8 spikes/4 s; after NPY, 23.1 ± 6.4 spikes/4 s; P < 0.01, two-tailed paired t-test), while the other antidromically activated neuron showed no change in firing rate following NPY injection (before NPY, 0.14 spikes/4 s; after NPY, 0 spikes/4 s). The NPY-induced increases in neuronal firing rate were accompanied by decreases in BAT SNA (Figure 5B; before NPY, 744.3 ± 123.7% baseline; after NPY, 29.5 ± 7.6% baseline; P < 0.001). The average antidromic latency and estimated conduction velocity of these NPY-activated neurons were 12.5 ± 0.9 ms and 0.22 ± 0.02 m/s, respectively. Two of these neurons exhibited obvious phasic bursting patterns after NPY injection (Figure 5C).

Figure 5. NPY-Mediated Neural Signaling Activates GABAergic IRt/PCRt Neurons Innervating the rMR.

Figure 5

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(A–C) In vivo extracellular unit recording of action potentials of a rat IRt/PCRt neuron. A collision test (A) shows an axonal projection of the IRt/PCRt neuron to the rMR. Single pulse stimulation in the rMR (triangles) evoked a constant-onset latency (6.7 ms) response in this neuron (arrow, top trace). rMR stimulation at 2.0 ms after a spontaneous action potential (open circle) failed to evoke a response of this neuron (middle trace). rMR stimulation at 10.0 ms after a spontaneous action potential evoked a constant-onset latency response of this neuron (bottom trace). All traces are superpositions of 3 stimulation trials. An intracerebroventricular (ICV) NPY injection ([B], arrowhead) increased the firing rate of this neuron (unit) and reversed BAT SNA and TBAT that had been increased by moderate body cooling. Obvious phasic bursting (C) was exhibited by some antidromically identified IRt/PCRt neurons following an ICV NPY injection.

(D) Histochemical identification of a recorded IRt/PCRt neuron innervating the rMR with juxtacellular labeling (green, arrow). In situ hybridization revealed that this neuron expressed Gad1 (GAD67) mRNA (red). Scale bar, 30 μm.

(E) Location of rMR-projecting IRt/PCRt neurons successfully identified with juxtacellular labeling. Among them, all neurons activated by NPY injection expressed Gad1 (filled circles), whereas the Gad1-negative one did not respond to NPY injection (open circle). Arrow indicates the neuron shown in (D).

Juxtacellular labeling following the unit recording was successful in 7 of the 11 antidromically activated IRt/PCRt neurons and confirmed their location in the IRt/PCRt (Figure 5D and E). To determine whether these labeled neurons were GABAergic, we performed in situ hybridization for mRNA of Gad1 (GAD67), a marker for GABAergic neurons. Six of the juxtacellularly labeled neurons expressed Gad1 and each of these was activated following the NPY injection, whereas the Gad1-negative neuron was the neuron unresponsive to NPY injection (Figure 5D and E). These results indicate that NPY-triggered neural signaling activates GABAergic IRt/PCRt neurons projecting to the rMR, supporting the idea that this inhibitory transmission mediates the NPY-induced inhibition of BAT thermogenesis and tachycardia.

Inactivation of IRt/PCRt Neurons Eliminates NPY-Induced Inhibition of BAT Thermogenesis and Tachycardia

To further examine the involvement of IRt/PCRt neurons in the hypothalamic NPY-induced sympathoinhibition, we tested the effect of inactivation of IRt/PCRt neurons on the hypothalamic NPY-induced inhibition of BAT thermogenic and tachycardic responses to skin cooling. Cooling the rat trunk skin consistently evoked BAT thermogenic, metabolic and tachycardic responses (Nakamura and Morrison, 2007). These responses were all suppressed by a unilateral injection of NPY, but not saline, into either the PVH or the lateral ventricle (Figures 6, S3 and S4). Importantly, this inhibitory effect of NPY was reversed by inactivation of IRt/PCRt neurons with bilateral nanoinjections of muscimol, but not by saline injections (Figures 6 and S4). These results are consistent with the view that the inhibitory transmission from GABAergic IRt/PCRt neurons to sympathetic premotor neurons in the rMR mediates NPY-triggered signaling from the hypothalamus to inhibit the central sympathetic drives for adaptive BAT thermogenesis and metabolism and for tachycardia. The GABAergic innervation of the rMR by IRt/PCRt neurons does not provide a tonic basal sympathoinhibition, because muscimol injections into the IRt/PCRt by themselves did not elicit BAT thermogenesis (Figure 6B).

Figure 6. Hypothalamic NPY-Induced Inhibition of BAT Thermogenesis, Metabolism and Tachycardia Requires Activation of IRt/PCRt Neurons.

Figure 6

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(A and B) Skin cooling (Tskin)-evoked increases in BAT thermogenesis, metabolism and HR were inhibited by NPY nanoinjection into the rat PVH following bilateral nanoinjections of saline into the IRt/PCRt (A), whereas bilateral nanoinjections of muscimol into the IRt/PCRt prevented the NPY-evoked inhibition (B).

(C and D) Effects of bilateral saline (C) and muscimol (D) nanoinjections into the IRt/PCRt on NPY-induced inhibition of skin cooling-evoked BAT thermogenic, metabolic and tachycardic responses. Skin cooling-evoked changes in physiological variables before and after saline or muscimol injections into the IRt/PCRt and NPY injection into the PVH are compared (n = 5). For data analysis, see Supplemental Experimental Procedures. ns: not significant, *P < 0.05, **P < 0.01 (paired t-test). All values are means ± SEM.

(E) Sites of injections into the PVH (left) and IRt/PCRt (right). For the IRt/PCRt, the left side of the symmetric bilateral injection sites is shown.

Stimulation of IRt/PCRt Neurons Evokes Mastication as well as Inhibits BAT Thermogenesis

Intriguingly, unilateral stimulation of IRt/PCRt neurons with a nanoinjection of bicuculline in anesthetized rats, which immediately reversed cooling-elevated BAT thermogenesis and heart rate, also evoked rhythmic masticatory movements of the masseter muscle with a frequency comparable to that observed following a central injection of NPY (Ushimura et al., 2015) (Figures 7A–C and S5, and Movie S1). The bicuculline injection also increased salivation in some animals. In free-moving rats, unilateral bicuculline injection into the IRt/PCRt promoted feeding behaviors with increased time spent chewing chow and bedding as well as increased food intake (Figure 7D and E), mimicking the feeding responses evoked by NPY injected into the PVH (Stanley and Leibowitz, 1985).

Figure 7. Stimulation of IRt/PCRt Neurons Promotes Mastication and Feeding As Well As Inhibits BAT Thermogenesis.

Figure 7

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(A–C) Unilateral nanoinjection of bicuculline into the IRt/PCRt in anesthetized rats (C) elicited mastication and inhibited cooling-evoked BAT thermogenesis and tachycardia (A). The masseter electromyogram (EMG) chart ([A], red horizontal bar) is expanded in (B). Figure S5B shows group data.

(D and E) Bicuculline injection into the IRt/PCRt in free-moving rats (E) increased chewing time and food intake measured for 1 hr after the injection (D) (saline: n = 5, bicuculline, n = 7). *P < 0.05 (Mann-Whitney test). All values are means ± SEM.

(F–H) Double retrograde labeling of GABAergic IRt/PCRt neurons with CTb injected into the Mo5 and with FluoroGold injected into the rMR (F) in an Slc32a1-Venus transgenic rat. Venus-expressing IRt/PCRt neurons were labeled with both CTb and FluoroGold ([G], arrows). Drawings (H) show the medullary distribution of CTb-labeled neurons and FluoroGold-labeled neurons with or without expression of Venus. Scale bars, 30 μm (G), 0.5 mm (H). DC, dorsal cochlear nucleus; Pn, pontine nuclei; Pr5, principal sensory trigeminal nucleus; scp, superior cerebellar peduncle; Su5, supratrigeminal nucleus.

(I) A model of the circuit that drives hunger responses. NPY-mediated signaling from the hypothalamus activates GABAergic IRt/PCRt neurons (red) to inhibit BAT thermogenesis. These GABAergic neurons facilitate rhythmic excitation of masticatory motoneurons potentially by interacting with glutamatergic neurons constituting the central pattern generator (CPG).

Thus, we examined the possibility that the GABAergic IRt/PCRt neurons projecting to the rMR are also involved in mastication. Slc32a1 (VGAT)-Venus transgenic rats, in which GABAergic neurons are labeled with Venus (Uematsu et al., 2008) (Figure S6), received injections with the retrograde tracers, FluoroGold and CTb, into the rMR and the motor trigeminal nucleus (Mo5), respectively (Figure 7F), the latter containing masticatory motoneurons (Mizuno et al., 1975). Many neurons double-labeled with FluoroGold and CTb were distributed in the IRt/PCRt at 2.6–2.8 mm caudal to the interaural line (Figure 7G and H), coincident with the region in which neural stimulation strongly inhibited BAT thermogenesis (Figure 3E). Importantly, 73% of these double-labeled neurons (151 of 206 cells) in the IRt/PCRt expressed Venus (Figure 7G and H). This observation supports the view that the axons of GABAergic IRt/PCRt neurons bifurcate to masticatory motoneurons and to BAT sympathetic premotor neurons, thereby providing the circuit mechanism for NPY-mediated hunger signaling from the hypothalamus to coordinate the promotion of mastication and feeding and the inhibition of adaptive thermogenesis (Figure 7I).

GABAergic IRt/PCRt Neurons Projecting to the rMR Receive Putative Synaptic Connections from the NTS and LPB

The PVH provides massive projections to the NTS, but much fewer to the IRt/PCRt (Shammah-Lagnado et al., 1992; Geerling et al., 2010; unpublished observation by Y.N. and K.N.). This observation raises the possibility that the BAT sympathoinhibitory, GABAergic IRt/PCRt neurons receive NPY-triggered signals from the hypothalamus by way of the NTS. Supporting this possibility, anterograde tracing with PHA-L injected into the NTS and retrograde tracing with FluoroGold injected into the rMR in Slc32a1-Venus transgenic rats showed close association of NTS-derived axon swellings with cell bodies of GABAergic IRt/PCRt neurons projecting to the rMR (Figure S7A and B).

The IRt/PCRt also receives projections, albeit fewer, from the lateral parabrachial nucleus (LPB) (Shammah-Lagnado et al., 1992), which has been proposed to receive a hunger signal from the PVH (Garfield et al., 2015). Injections of PHA-L and FluoroGold into the LPB and rMR, respectively, in Slc32a1-Venus transgenic rats visualized LPB-derived axon swellings apposed to cell bodies of GABAergic IRt/PCRt neurons projecting to the rMR (Figure S7C and D). However, PHA-L-labeled fibers from the LPB were more sparsely distributed in the IRt/PCRt than those from the NTS. These anatomical observations indicate that GABAergic IRt/PCRt neurons innervating the rMR can receive inputs from the NTS and LPB (Figure 7I).

DISCUSSION

Reduced energy expenditure and stimulated appetite are fundamental innate strategies that mammals use to survive starvation, and may, perhaps to a lesser extent, be employed in normal feeding cycles. Many studies have focused on the hypothalamic circuit that provides command signals to elicit the physiological and behavioral responses upon receiving hunger signals from peripheral organs, such as ghrelin (Loh et al., 2015). In this circuit, NPY is well known as a powerful hypothalamic neuropeptide that can trigger the hunger responses (Loh et al., 2015). However, the circuit mechanism by which the commands from the hypothalamic network affect the medullary motor systems to alter autonomic thermogenesis and feeding motor functions has been poorly understood. The present study highlights the IRt/PCRt as an important component in the hypothalamomedullary hunger response circuit. We showed that GABAergic neurons in the IRt/PCRt provide direct axonal projections to the rMR with putative synaptic connections to BAT sympathetic premotor neurons, and that activation of neurons in the limited area of the IRt/PCRt eliminates BAT thermogenesis and tachycardia evoked by skin cooling or pyrogenic stimulation. Furthermore, our chemogenetic experiments demonstrated that a GABAergic population of IRt/PCRt neurons is responsible for the sympathoinhibitory effects. These findings indicate that GABAergic IRt/PCRt neurons can suppress BAT thermogenesis and tachycardia through their inhibitory inputs to sympathetic premotor neurons in the rMR.

In support of this view, stimulation of IRt/PCRt neurons failed to inhibit BAT thermogenesis and tachycardia evoked by disinhibition of sympathetic premotor neurons effected by antagonizing GABAA receptors in the rMR. Similarly, NPY injection into the PVH, which inhibited BAT thermogenesis and cardiovascular responses to glutamatergic stimulation of sympathetic premotor neurons in the rMR, failed to inhibit the thermogenic and cardiovascular responses evoked by antagonizing GABAA receptors in the rMR. These results indicate that hypothalamic NPY-triggered neural signaling also inhibits BAT thermogenesis and tachycardia through GABAergic inhibition of sympathetic premotor neurons in the rMR. Consistently, the present in vivo unit recording followed by in situ hybridization showed that NPY-triggered neural signaling activates GABAergic transmission from the IRt/PCRt to the rMR. Furthermore, inactivation of IRt/PCRt neurons blocked the inhibitory effects of hypothalamic NPY on BAT thermogenesis and tachycardia. All these results support the view that NPY-mediated neural signaling from the hypothalamus in response to negative energy balance activates GABAergic transmission from the IRt/PCRt to sympathetic premotor neurons in the rMR to suppress the excitatory outflows driving active BAT thermogenesis and tachycardia for cold defense and the development of fever (Figure 7I). This mechanism is consistent with the physiological action of central NPY that inhibits active heat production in sub-thermoneutral environments, but not basal metabolic rate (Székely et al., 2005).

The present study revealed that many GABAergic IRt/PCRt neurons innervating the BAT sympathetic premotor region (rMR) also innervate the masticatory motor region (Mo5). Indicating the involvement of IRt/PCRt neurons in the control of both mastication and metabolism, stimulation of IRt/PCRt neurons elicited mastication as well as an inhibition of BAT thermogenesis. Surprisingly, stimulation of IRt/PCRt neurons in free-moving rats increased chewing and feeding, similar to the action of hypothalamic NPY (Stanley and Leibowitz, 1985; Ushimura et al., 2015). Supporting the critical function of the IRt/PCRt in the orexigenic action of NPY, inactivation of IRt/PCRt neurons reduces mastication and feeding elicited by hypothalamic NPY (Travers et al., 2010). The IRt/PCRt contains masticatory premotor neurons, some of which are GABAergic (Li et al., 1996; Travers et al., 2005), and constitutes the central pattern generator for the rhythmic masticatory muscle movements (Moore et al., 2014). Although the circuit mechanism of the central pattern generator has yet to be determined, both glutamatergic and GABAergic premotor inputs from the IRt/PCRt to the Mo5 are likely activated for the generation of the rhythmic masticatory motor activities (Nakamura and Katakura, 1995) including the NPY-induced mastication. Phasic bursting patterns similar to masticatory rhythm were observed in our unit recording from GABAergic, rMR-projecting IRt/PCRt neurons (Figure 5C), suggesting the involvement of these IRt/PCRt neurons in the NPY-induced mastication (Figure 7I).

The NPY-triggered masticatory premotor signaling from the IRt/PCRt likely primes the motor system to be “ready to eat”, and the final motor output may be gated by corticomedullary inputs (Nakamura and Katakura, 1995; Moore et al., 2014), which could be triggered by visual and olfactory sensation of food, to initiate mastication dependent on food availability. Under the anesthetized condition in the present study, the mastication we observed following stimulation of IRt/PCRt neurons was likely due to an anesthesia-mediated reduction of such a top-down control of mastication from the cerebral cortex. On the other hand, our selective chemogenetic stimulation of GABAergic neurons in the IRt/PCRt did not elicit masticatory movements (unpublished observation by Y.N. and K.N.), probably because the masticatory motor rhythm generation requires activation of both GABAergic and glutamatergic premotor neurons in the IRt/PCRt. The paucity of non-GABAergic IRt/PCRt neurons projecting to both the rMR and Mo5 (Figure 7H) suggests that GABAergic masticatory premotor neurons in the IRt/PCRt, rather than glutamatergic ones, mediate the NPY-triggered inhibition of BAT thermogenesis through their axon collaterals to the rMR (Figure 7I).

Although it is still uncertain how the NPY-mediated signal is transmitted from the PVH to the IRt/PCRt, the PVH provides massive projections to the NTS (Geerling et al., 2010), and our tracing study revealed putative synaptic connections of axons from the NTS to GABAergic IRt/PCRt neurons innervating the rMR (Figure S7). Furthermore, stimulation of rostral NTS neurons inhibited BAT thermogenesis (Figure 3E). The insignificant number of GABAergic neurons in the rostral NTS that project to the rMR in either mice or rats (Figures 2C and 7H) indicates negligible monosynaptic inhibitory innervation from the NTS to the rMR. Therefore, NTS neurons could mediate the NPY-triggered signaling from the hypothalamus to GABAergic IRt/PCRt neurons to indirectly inhibit sympathetic premotor neurons in the rMR. Because the NTS receives a variety of vagal visceral afferents, some of which may inhibit BAT SNA (Morrison et al, 2014; Madden et al, 2016), the hypothalamic NPY signal could be integrated with nutritional visceral information impinging on NTS neurons before being transmitted to the IRt/PCRt (Figure 7I).

We also found that GABAergic IRt/PCRt neurons innervating the rMR receive putative synaptic inputs from the LPB. The LPB, but not the NTS, receives excitatory inputs from melanocortin-4 receptor (MC4R)-expressing neurons in the PVH and optogenetic stimulation of this transmission to the LPB alters food intake (Garfield et al., 2015). NPY/AgRP/GABA-containing axons from the arcuate nucleus provide synaptic inputs to 83% of MC4R-expressing neurons and 20% of non-MC4R neurons in the PVH (Garfield et al., 2015). Although the present study only examined the NPY-triggered hunger response signaling, AgRP, co-released from arcuate NPY neurons, can decrease metabolism and increase feeding by inhibiting the melanocortin system through antagonizing MC4Rs in the PVH (Cowley et al., 1999; Sohn et al., 2013). The responses induced by AgRP are likely mediated by neural pathways different from those mediating the NPY-triggered hunger responses (Krashes et al., 2013). Together with our present finding, therefore, AgRP released in the PVH may trigger a hunger response signaling through the PVH–LPB–IRt/PCRt pathway by antagonizing melanocortin inputs to MC4R-expressing PVH neurons. In this hypothesis, whether GABAergic IRt/PCRt neurons mediate the AgRP-triggered signaling is an attractive question awaiting further investigation. On the other hand, NPY might act on non-MC4R PVH neurons, potentially oxytocin neurons (Atasoy et al., 2012), to stimulate hunger response signaling through the PVH–NTS–IRt/PCRt pathway. It should be noted that NPY receptor-expressing PVH neurons can receive NPY through its volume transmission even if they do not all receive NPY/AgRP neuron synaptic inputs.

Whether NPY activates or inhibits PVH neurons is controversial (Mercer et al., 2011). NPY activates (disinhibits) PVH neurons by inhibiting GABA release from terminals that synapse on parvocellular PVH neurons (Cowley et al., 1999; Pronchuk et al., 2002), and central NPY injection induces expression of Fos, a marker of neuronal activation, in PVH neurons (Li et al., 1994). Similar to NPY injection into the PVH, non-selective stimulation of PVH neurons with NMDA or bicuculline inhibits BAT thermogenesis evoked by glutamatergic stimulation of the rMR, but not that evoked by antagonizing GABAA receptors in the rMR (Madden and Morrison, 2009). This similarity of the effects of NPY and the non-selective neural stimulation in the PVH supports the view that activation of PVH neurons by NPY gives rise to the descending signal that activates the IRt/PCRt–rMR GABAergic transmission to inhibit BAT thermogenesis. On the other hand, NPY also postsynaptically inhibits the discharge of MC4R-expressing PVH neurons (Ghamari-Langroudi et al., 2011). Optogenetic stimulation of NPY/AgRP/GABA neuronal transmission from the arcuate nucleus to the PVH evokes inhibitory postsynaptic currents in PVH neurons and also elicits a hyperphagic response that is reduced by antagonizing either NPY or GABAA receptors in the PVH (Atasoy et al., 2012). If inhibition of PVH neurons by NPY leads to the stimulation of the IRt/PCRt–rMR GABAergic transmission, the pathway from the PVH to the IRt/PCRt would involve a disinhibitory mechanism, which awaits further investigation.

NPY-containing neurons are also distributed in the dorsomedial hypothalamus (DMH) in addition to the arcuate nucleus and knockdown of NPY expression in the DMH increases energy expenditure and thermogenic responses to cold (Chao et al., 2011), suggesting a role of DMH-derived NPY in reducing metabolism and BAT thermogenesis. The projections of NPY-containing DMH neurons are mostly limited to hypothalamic regions including the PVH (Lee et al., 2013). In the present study, NPY injection into the lateral ventricle, as well as into the PVH, strongly inhibited skin cooling-evoked BAT thermogenesis and tachycardia, and, independent of injection site, the inhibitory effects of NPY were completely reversed by inactivation of IRt/PCRt neurons. This result indicates that IRt/PCRt neurons mediate NPY-triggered sympathoinhibitory signaling not only from the PVH, but also from any other hypothalamic sites at which NPY may act.

The IRt/PCRt has never been associated with the control of thermogenesis or metabolism. The present study demonstrates that GABAergic neurons in this medullary reticular formation function in the hypothalamomedullary hunger response signaling to inhibit sympathetic premotor outflows to BAT and the heart to reduce energy expenditure for the survival of starvation. Our results also show that these GABAergic neurons can contribute to the generation of masticatory premotor activities to enable the animals to initiate food intake as soon as it becomes available. These findings clearly indicate that the IRt/PCRt coordinates the autonomic and somatic motor systems in response to the hunger signaling from the hypothalamus to drive the metabolic and consummatory hunger responses (Figure 7I). Hyperactivity of the GABAergic IRt/PCRt neurons under non-starved conditions likely contributes to the development of pathological states leading to obesity. The inputs and the mechanisms that regulate the activity of GABAergic IRt/PCRt neurons will be a next focus of interest.

EXPERIMENTAL PROCEDURES

Animals

Male Sprague-Dawley (SD) rats (200–500 g), male Slc32a1-Venus (VGAT-Venus-B) transgenic rats (also known as Tg(Slc32a1-YFP*)2Yyan; 250–500 g; Uematsu et al., 2008), male Gad1-Gfp (GAD67-GFP) knock-in mice (also known as Gad1tm1.1Tama; 25–40 g; Tamamaki et al., 2003) and male Gad2-IRES-Cre (GAD65-ires-Cre) knock-in mice (also known as Gad2tm2(cre)Zjh; 25–30 g; Jackson Laboratory stock 010802; Taniguchi et al., 2011) were used. They were housed two or three to a cage with ad libitum access to food and water in a room air-conditioned at 24 ± 2°C with a standard 12 h light/dark cycle before used for surgery or experiments. All procedures conform to the guidelines of animal care by the Division of Experimental Animals, Nagoya University Graduate School of Medicine and to the regulations detailed in the National Institutes of Health Guide for the Care and Use of Laboratory Animals, and were approved by the Nagoya University Animal Experiment Committee and by the Animal Care and Use Committee of the Oregon Health & Science University.

Neural tract tracing

Animals received injections of Alexa594-conjugated CTb (Invitrogen), FluoroGold (Fluorochrome) or PHA-L (Vector) into a specific brain site under deep anesthesia (see Supplemental Experimental Procedures). Four to eight days after the tracer injection, the animals were re-anesthetized and transcardially perfused with saline and then with 4% (or 2% for VGLUT3 immunohistochemistry) formaldehyde in 0.1 M phosphate buffer (pH 7.4). The brain was removed, postfixed in the fixative at 4°C for 2–3 hrs, and then cryoprotected in a 30% sucrose solution overnight. The tissue was cut into 30-μm-thick (rat) or 20-μm-thick (mouse) frontal sections on a freezing microtome. Data from animals where tracer injection was not centered in the target brain region were excluded. CTb and PHA-L were detected in the brain sections with immunohistochemical staining and FluoroGold was detected with the fluorescence of the tracer.

Immunohistochemistry

Immunohistochemistry followed our methods (Nakamura et al., 2004, 2005; see Supplemental Experimental Procedures). The primary antibodies used are anti-CTb goat serum (#703, List Biological Laboratories), anti-GFP mouse antibody (A11120, Life Technologies), anti-GFP guinea pig serum (Tamamaki et al., 2000), anti-mRFP rabbit antibody (Hioki et al., 2010), anti-PHA-L rabbit antibody (AL-1801-2, EY laboratories), anti-PHA-L goat antibody (AS-2224, Vector), anti-VGLUT3 guinea pig antibody (Hioki et al., 2004), and anti-VGAT rabbit antibody (VGAT11-A, Alpha Diagnostics). The anti-GFP mouse antibody and the anti-mRFP antibody showed reactivity to Venus and mCherry, respectively.

In situ hybridization

In situ hybridization for Gad1 mRNA was performed according to our previous method (Nakamura et al., 2002), except the hybridization signals were visualized with Fast Red according to manufacturer’s guide (Roche). For subsequent immunofluorescence labeling of Venus, the sections were incubated with an anti-heat-denatured GFP rabbit antibody (1 μg/ml; Nakamura et al., 2008). The immunoreactivity was visualized by incubating the sections with a biotinylated donkey antibody to rabbit IgG (1:100; AP182B, Millipore) and then with 2 μg/ml Alexa488-conjugated streptavidin (S11223, Invitrogen).

In vivo electrophysiology in anesthetized rats

The procedure for in vivo physiological recordings basically followed our previous studies (Nakamura and Morrison, 2007, 2011; see Supplemental Experimental Procedures). In SD rats anesthetized with urethane and α-chloralose, postganglionic BAT SNA was electrophysiologically recorded from a nerve bundle isolated from the right interscapular BAT pad. Temperature of the left interscapular BAT pad, rectal temperature, trunk skin temperature, mixed expired CO2, arterial pressure, and heart rate were also recorded simultaneously. In some animals, electromyogram (EMG) was also recorded with bipolar needle electrodes inserted into the masseter muscle.

In vivo unit recording was performed as described (Nakamura and Morrison, 2008, 2010) with a recording glass microelectrode filled with 0.5 M sodium acetate (DC resistance: 18–39 MΩ) containing 5% biotinamide to allow juxtacellular labeling of the recorded neurons. A monopolar tungsten stimulating microelectrode was stereotaxically positioned in the rMR. The IRt/PCRt was explored with the recording electrode for neurons showing a constant onset latency response to electrical stimulation in the rMR (0.2–3 mA, 1 ms, 0.25–0.33 Hz). Standard criteria (Morrison and Cao, 2000) were used to establish the antidromic nature of the responses of IRt/PCRt neurons to rMR stimulation. After time-controlled collision tests (Figure 5A), the effects of an NPY injection into the lateral ventricle (1 mg/ml, 5 μl) on the spontaneous firing rate of the antidromically identified neuron and on cooling-increased BAT SNA and BAT temperature were tested. For juxtacellular labeling (Pinault, 1996) of the recorded neurons with biotinamide, positive current pulses (0.5–4.0 nA, 200 ms duration, 50% duty cycle) were delivered for 1–5 min to entrain the neuron. After the recording, the animals were perfused with 4% formaldehyde and the labeled neurons were visualized by incubating the brain sections with 1 μg/ml Alexa488-conjugated streptavidin. The sections were further processed for in situ hybridization to detect Gad1 mRNA expression as above. For firing rate analysis of each recorded neuron, the rate of spontaneous firing was measured as spikes/4 s and averaged for 30 s immediately before NPY injection and after NPY-induced inhibition of BAT SNA to compare.

In vivo chemogenetic experiment in anesthetized mice

Gad2-IRES-Cre knock-in mice received bilateral injections into the IRt/PCRt with AAV-EF1α-DIO-hM3Dq-mCherry. More than 1 week later, the mice were anesthetized with urethane, and their interscapular BAT temperature, abdominal skin temperature, rectal temperature and heart rate (converted from electrocardiogram) were monitored (see Supplemental Experimental Procedures). CNO (0.1 mg/ml, dissolved in 0.5% DMSO), vehicle (0.5% DMSO in saline) or 2 mM muscimol was injected bilaterally into the IRt/PCRt (30 nl/site) and subsequently, the abdominal skin was cooled and rewarmed repetitively by using a water jacket. The effects of the injectates on skin cooling-induced BAT thermogenesis and tachycardia were tested in the order of vehicle, CNO and muscimol.

Bicuculline injection into the IRt/PCRt in free-moving rats

SD rats received a unilateral nanoinjection of 1 mM bicuculline or saline (50–100 nl) into the IRt/PCRt through a pre-implanted cannula. Food intake and chewing time were measured in feeding monitoring cages (Feedam, Melquest) for 1 hr after the injection. For detailed procedure, see Supplemental Experimental Procedures.

Anatomy and Statistical Analysis

We adopted the cytoarchitecture and nomenclature of most brain regions from those of Paxinos and Watson (1998) for rats and Franklin and Paxinos (2007) for mice. The raphe pallidus nucleus was nomenclaturally divided into two parts, rostral (rRPa) and caudal (cRPa), at the rostral end of the inferior olivary complex (Nakamura et al., 2002, 2004).

Data are shown as the means ± SEM. Statistic comparison analyses were performed using a paired or unpaired t-test, Mann-Whitney test, a repeated measures one-way ANOVA followed by Tukey’s multiple comparisons test, or a two-way ANOVA followed by Bonferroni’s multiple comparisons test (Prism 6, GraphPad) as stated in the text and figure legends. All the statistic tests were two-sided. P < 0.05 was considered statistically significant.

Supplementary Material

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Download video file (20.7MB, mov)

Acknowledgments

We thank Chika Tanizawa and Miki Iwatani for anatomical assistance, Naoya Kataoka for providing AAV, Hiroyuki Hioki for providing anti-VGLUT3, anti-GFP, anti-denatured GFP and anti-mRFP antibodies, and Atsushi Miyawaki for providing pCS2-Venus. This study was supported by the Funding Program for Next Generation World-Leading Researchers from the Japan Society for the Promotion of Science (LS070 to K.N.), by Grants-in-Aid for Scientific Research (16H05128, 15H05932, 15K21744, 26118508 and 26713009 to K.N., 26290002 and 15H01415 to Y.Y., and 26860159 to Y.N.) and a Grant-in-Aid for Scientific Research on Innovative Areas (Comprehensive Brain Science Network) (to Y.Y.) from the MEXT of Japan, by the U.S. NIH grant (R01NS40987 and R01NS91066 to S.F.M.), and by grants from the Takeda Science Foundation (to K.N. and Y.Y.), Nakajima Foundation, Uehara Memorial Foundation, Brain Science Foundation, and Kowa Life Science Foundation (to K.N.).

Footnotes

AUTHOR CONTRIBUTIONS

Y.N., S.F.M and K.N. designed and performed experiments. Y.N. and K.N. analyzed data. Y.Y. provided Gad1-Gfp knock-in mice and Slc32a1-Venus transgenic rats. Y.N., S.F.M and K.N. discussed data and wrote the manuscript, and all authors approved the manuscript.

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