Shigefumi Yokota

Neuroanatomical and neurochemical organization of projections from the central amygdaloid nucleus to the nucleus retroambiguus via the periaqueductal gray in the rat.

The periaqueductal gray (PAG)-nucleus retroambiguus (NRA) pathway has been known to be involved in the control of vocalization and sexual behavior. To know how the amygdaloid complex influences the PAG-NRA pathway, here we first examined the synaptic organization between the central amygdaloid nucleus (CeA) fibers and the PAG neurons that project to the NRA by using anterograde and retrograde tract-tracing techniques in the rat. After ipsilateral injections of biotinylated dextran amine (BDA) into the CeA and cholera toxin B subunit (CTb) into the NRA, the prominent overlapping distribution of BDA-labeled axon terminals and CTb-labeled neurons was found ipsilaterally in the lateral/ventrolateral PAG, where some of the BDA-labeled terminals made symmetrical synaptic contacts with somata and dendrites of the CTb-labeled neurons. After CTb injection into the lateral/ventrolateral PAG, CTb-labeled neurons were distributed mainly in the medial division of the CeA. After BDA injection into the lateral/ventrolateral PAG, BDA-labeled fibers were distributed mainly in and around the NRA within the medulla oblongata. Using a combined retrograde tracing and in situ hybridization technique, we further demonstrated that more than half of the CeA neurons labeled with Fluoro-Gold (FG) injected into the lateral/ventrolateral PAG were positive for glutamic acid decarboxylase 67 mRNA and that the vast majority of PAG neurons labeled with FG injected into the NRA expressed vesicular glutamate transporter 2 mRNA. The present results suggest that the glutamatergic PAG-NRA pathway is under the inhibitory influence of the GABAergic CeA neurons.

Amygdaloid axons innervate melanin-concentrating hormone-and orexin-containing neurons in the mouse lateral hypothalamus

This study was performed to understand the anatomical substrates of amygdaloid modulation of feeding-related peptides-containing neurons in the lateral hypothalamic area (LHA). After biotinylated dextranamine (BDA) injection into the central amygdaloid nucleus (CeA) and immunostaining of melanin-concentrating hormone (MCH)- or orexin (ORX)-containing hypothalamic neurons in the mouse, the prominent overlap of the distribution field of the BDA-labeled fibers and that of the MCH-immunoreactive (ir) or ORX-ir neurons was found in the dorsolateral part of the LHA, and the labeled axon terminals made symmetrical synaptic contacts with somata and dendrites of the MCH-ir or ORX-ir neurons. It was further revealed that nearly all the BDA-labeled axon terminals in the dorsolateral part of LHA were immunoreactive for glutamic acid decarboxylase, an enzyme for conversion of glutamic acid to gamma-aminobutyric acid (GABA). The present data suggest that the CeA is involved in the regulation of feeding behavior by exerting its GABAergic inhibitory action upon the MCH- and ORX-containing LHA neurons.

Spatiotemporal and anatomical analyses of P2X receptor-mediated neuronal and glial processing of sensory signals in the rat dorsal horn

Summary Adenosine triphosphate in the dorsal horn acts on neuronal pre‐ and postsynaptic P2X receptors, especially in the deep layer, and astrocytes optimize the excitability, especially in the superficial layer. ABSTRACT Extracellularly released adenosine triphosphate (ATP) modulates sensory signaling in the spinal cord. We analyzed the spatiotemporal profiles of P2X receptor‐mediated neuronal and glial processing of sensory signals and the distribution of P2X receptor subunits in the rat dorsal horn. Voltage imaging of spinal cord slices revealed that extracellularly applied ATP (5–500 μM), which was degraded to adenosine and acting on P1 receptors, inhibited depolarizing signals and that it also enhanced long‐lasting slow depolarization, which was potentiated after ATP was washed out. This post‐ATP rebound potentiation was mediated by P2X receptors and was more prominent in the deep than in the superficial layer. Patch clamp recording of neurons in the superficial layer revealed long‐lasting enhancement of depolarization by ATP through P2X receptors during the slow repolarization phase at a single neuron level. This depolarization pattern was different from that in voltage imaging, which reflects both neuronal and glial activities. By immunohistochemistry, P2X1 and P2X3 subunits were detected in neuropils in the superficial layer. The P2X5 subunit was found in neuronal somata. The P2X6 subunit was widely expressed in neuropils in the whole gray matter except for the dorsal superficial layer. Astrocytes expressed the P2X7 subunit. These findings indicate that extracellular ATP is degraded into adenosine and prevents overexcitation of the sensory system, and that ATP acts on pre‐ and partly on postsynaptic neuronal P2X receptors and enhances synaptic transmission, predominantly in the deep layer. Astrocytes are involved in sensitization of sensory network activity more importantly in the superficial than in the deep layer.

Glutamatergic lateral parabrachial neurons innervate orexin-containing hypothalamic neurons in the rat.

We performed this study to understand the anatomical substrates of parabrachial nucleus (PBN) modulation of orexin (ORX)-containing neurons in the hypothalamus. After biotinylated dextranamine (BDA) injection into the lateral PBN and immunostaining of ORX-containing neurons in the rat, the prominent overlap of the distribution field of the BDA-labeled fibers and that of the ORX-immunoreactive (ir) neurons was found in the lateralmost part of the dorsomedial nucleus and adjacent dorsal perifornical area (this overlapping field was referred to as suprafornical area in the present study), and the labeled axon terminals made asymmetrical synaptic contacts with somata and dendrites of the ORX-ir neurons. We further revealed that almost all the suprafornical area-projecting lateral PBN neurons were positive for vesicular glutamate transporter 2 mRNA and very few of them were positive for glutamic acid decarboxylase 67 mRNA. The present data suggest that ORX-containing neurons in the suprafornical area may be under the excitatory influence of the glutamatergic lateral PBN neurons probably for the regulation of arousal and waking.

Glutamatergic Kölliker–Fuse nucleus neurons innervate hypoglossal motoneurons whose axons form the medial (protruder) branch of the hypoglossal nerve in the rat

This study was performed to understand the anatomical substrates for Kölliker-Fuse nucleus (KFN) modulation of respiratory-related tongue movement. After application of cholera toxin B subunit (CTb) to the medial branch of the hypoglossal nerve (HGn) and injection of biotinylated dextran amine (BDA) into the KFN ipsilaterally, an overlapping distribution of BDA-labeled axon terminals and CTb-labeled neurons was found in the ventral compartment of the hypoglossal nucleus (HGN) ipsilateral to the application and injection sites. At the electron microscopic level, the BDA-labeled terminals made asymmetrical synaptic contacts predominantly with dendrites of the HGN neurons, some of which were labeled with CTb. Using retrograde tracing combined with in situ hybridization, we demonstrated that almost all the KFN neurons sending their axons to the HGN were positive for vesicular glutamate transporter (VGLUT) 2 mRNA but not glutamic acid decarboxylase 67 mRNA. Using a combination of anterograde and retrograde tracing techniques and immunohistochemistry for VGLUT2, we further demonstrated that the KFN axon terminals with VGLUT2 immunoreactivity established close contact with the HGN motoneurons whose axons constitute the medial branch of the HGn. The present results suggest that glutamatergic KFN fibers may exert excitatory influence upon the HGN motoneurons sending their axons to the medial branch of the HGn for the control of protruder tongue muscles contraction to maintain airway patency during respiration.

A disynaptic pathway from the central amygdaloid nucleus to the paraventricular hypothalamic nucleus via the parastrial nucleus in the rat

The organization of projections from the central amygdaloid nucleus (CeA) to the paraventricuilar hypothalamic nucleus (PVH) has been studied in order to understand the anatomical substrates of amygdaloid modulation of endocrine and autonomic functions, and a hypothesis that the bed nucleus of the stria terminalis (BST) may act as a relay site between the CeA and PVH has been proposed. Using anterograde and retrograde tract-tracing methods, in the rat, we first indicated that neurons in the parastrial nucleus (PS), where projection fibers from the central amygdaloid nucleus (CeA) terminated, sent their axons to the paraventricular hypothalamic nucleus (PVH). We further demonstrated that the CeA terminals formed symmetrical synaptic contacts with somata and dendrites of the PVH-projecting PS neurons, and that the PS received CeA fibers predominantly from the lateral part and sent large numbers of projection fibers to almost all the subdivisions of the PVH. Using anterograde tracing combined with the postembedding immunogold method, we finally revealed that nearly all the CeA terminals in the PS were immunoreactive for gamma-aminobutyric acid. The present data suggest that output signals from the CeA are transmitted disynaptically to the PVH neurons via the PS neurons and modulate PVH neuron activity by way of disinhibition.

Posterior lateral hypothalamic axon terminals are in contact with trigeminal premotor neurons in the parvicellular reticular formation of the rat medulla oblongata.

This study was performed to understand the anatomical substrates of hypothalamic modulation of jaw movements. After cholera toxin B subunit (CTb) injection into the parvicellular reticular formation (RFp) of the rat medulla oblongata, where many trigeminal premotor neurons have been known to exist, numerous CTb-labeled neurons were found in the posterior lateral hypothalamus (PLH) bilaterally with a clear-cut ipsilateral dominance. After ipsilateral injections of biotinylated dextran amine (BDA) into the PLH and CTb into the motor trigeminal nucleus (Vm), the prominent distribution of BDA-labeled axon terminals around CTb-labeled neurons was found in the RFp region just ventral to the nucleus of the solitary tract and medial to the spinal trigeminal nucleus ipsilateral to the injection sites. Within the neuropil of the RFp, BDA-labeled axon terminals made an asymmetrical synaptic contact predominantly with dendrites and additionally with somata of the RFp neurons, some of which were labeled with CTb. It was further revealed that these BDA-labeled axon terminals were immunoreactive for vesicular glutamate transporter 2. The present data suggest that the PLH plays an important role in the control of jaw movements by exerting its glutamatergic excitatory action upon RFp neurons presynaptic to trigeminal motoneurons.

GABAergic neurons in the ventrolateral subnucleus of the nucleus tractus solitarius are in contact with Kölliker-Fuse nucleus neurons projecting to the rostral ventral respiratory group and phrenic nucleus in the rat

After ipsilateral injections of biotinylated dextran amine (BDA) into the ventrolateral subnucleus of the nucleus tractus solitarius (vlNTS) and Fluoro-gold (FG) into the rostral ventral respiratory group (rVRG) region or into the phrenic nucleus (PhN) region in the rat, an overlapping distribution of BDA-labeled axon terminals and FG-labeled neurons was found in the Kölliker-Fuse (KF) nucleus ipsilateral to the injection sites. Using retrograde tracing combined with immunohistochemistry for glutamic acid decarboxylase isoform 67 (GAD67), we indicated that as many as 40% of the vlNTS neurons projecting to the KF were immunoreactive for GAD67. Using a combination of anterograde and retrograde tracing techniques, and immunohistochemistry for GAD67, we further demonstrated that the vlNTS axon terminals with GAD67 immunoreactivity established close contact to the rVRG- or PhN-projecting KF neurons. The present results suggest that GABAergic vlNTS fibers may exert inhibitory influences on the rVRG- as well as PhN-projecting KF neurons and these circuits may be involved in the respiratory reflexes such as the Hering-Breuer reflex.

Central amygdaloid axon terminals are in contact with retrorubral field neurons that project to the parvicellular reticular formation of the medulla oblongata in the rat

The retrorubral field (RRF) contains numerous dopaminergic neurons and projects to the parvicellular reticular formation (RFp) of the medullary and pontomedullary brainstem, where many premotor neurons project to the orofacial motor nuclei. To know how the amygdala affects the RRF-RFp pathway in the rat, we first examined the synaptic organization between the central amygdaloid nucleus (CeA) fibers and the RFp-projecting RRF neurons by using combined anterograde and retrograde tracing techniques. After ipsilateral injections of biotinylated dextran amine (BDA) into the CeA and Fluoro-gold (FG) into the RFp, the prominent overlapping distribution of BDA-labeled axon terminals and FG-labeled neurons was found in the lateral part of the RRF ipsilateral to the injection sites, where the BDA-labeled axon terminals made symmetrical synapses with somata and dendrites of the FG-labeled neurons. Using a combination of retrograde tracing and immunohistochemistry for tyrosine hydroxylase (TH), we secondly demonstrated that the RFp-projecting RRF neurons were immunonegative for TH. Using a combination of anterograde tracing and immunohistochemistry for glutamic acid decarboxylase (GAD), we finally revealed that the CeA axon terminals in the RRF were immunoreactive for GAD. The present results suggest that GABAergic CeA neurons may exert inhibitory influences on non-dopaminergic RRF neurons that project to the RFp in the control of orofacial movements closely related to emotional behavior.

Anatomical Architecture and Responses to Acidosis of a Novel Respiratory Neuron Group in the High Cervical Spinal Cord (HCRG) of the Neonatal Rat

It has been postulated that there exists a neuronal mechanism that generates respiratory rhythm and modulates respiratory output pattern in the high cervical spinal cord. Recently, we have found a novel respiratory neuron group in the ventral portion of the high cervical spinal cord, and named it the high cervical spinal cord respiratory group (HCRG). In the present study, we analyzed the detailed anatomical architecture of the HCRG region by double immunostaining of the region using a neuron-specific marker (NeuN) and a marker for motoneurons (ChAT) in the neonatal rat. We found a large number of small NeuN-positive cells without ChAT-immunoreactivity, which were considered interneurons. We also found two and three clusters of motoneurons in the ventral portion of the ventral horn at C1 and C2 levels, respectively. Next, we examined responses of HCRG neurons to respiratory and metabolic acidosis in vitro by voltage-imaging together with cross correlation techniques, i.e., by correlation coefficient imaging, in order to understand the functional role of HCRG neurons. Both respiratory and metabolic acidosis caused the same pattern of changes in their spatiotemporal activation profiles, and the respiratory-related area was enlarged in the HCRG region. After acidosis was introduced, preinspiratory phase-dominant activity was recruited in a number of pixels, and more remarkably inspiratory phase-dominant activity was recruited in a large number of pixels. We suggest that the HCRG composes a local respiratory neuronal network consisting of interneurons and motoneurons and plays an important role in respiratory augmentation in response to acidosis.