Orexinergic fibers are in contact with Kölliker-Fuse nucleus neurons projecting to the respiration-related nuclei in the medulla oblongata and spinal cord of the rat.

The neural pathways underlying the respiratory variation dependent on vigilance states remain unsettled. In the present study, we examined the orexinergic innervation of Kölliker-Fuse nucleus (KFN) neurons sending their axons to the rostral ventral respiratory group (rVRG) and phrenic nucleus (PhN) as well as to the hypoglossal nucleus (HGN) by using a combined retrograde tracing and immunohistochemistry. After injection of cholera toxin B subunit (CTb) into the KFN, CTb-labeled neurons that are also immunoreactive for orexin (ORX) were found prominently in the perifornical and medial regions and additionally in the lateral region of the hypothalamic ORX field. After injection of fluorogold (FG) into the rVRG, PhN or HGN, we found an overlapping distribution of ORX-immunoreactive axon terminals and FG-labeled neurons in the KFN. Within the neuropil of the KFN, asymmetrical synaptic contacts were made between these terminals and neurons. We further demonstrated that many neurons labeled with FG injected into the rVRG, PhN, or HGN are immunoreactive for ORX receptor 2. Present data suggest that rVRG-, PhN- and HGN-projecting KFN neurons may be under the excitatory influence of the ORXergic neurons for the state-dependent regulation of respiration.

Intrapancreatic ganglia neurons receive projection fibers from melanocortin-4 receptor-expressing neurons in the dorsal motor nucleus of the vagus nerve of the mouse.

Melanocortin-4 receptor (MC4R)-expressing neurons are widely distributed in the central nervous system and play a crucial role in a variety of physiological functions including energy and glucose/insulin homeostasis. However, their neural pathways remain to be elucidated. In the present study, we examined a possible pathway from MC4R-expressing neurons in the dorsal motor nucleus of the vagus nerve (DMV) to the intrapancreatic ganglia using transgenic mice that express green fluorescent protein (GFP) under the control of the MC4R-promoter. Using immunofluorescence labeling, we demonstrated that GFP-immunoreactive (ir) nerve fibers were distributed in the intrapancreatic ganglia closely associated with the islets as well as among the acini. These GFP-ir fibers with bouton-like varicosities were frequently observed to surround ganglion cells immunoreactive for vasoactive intestinal polypeptide, a marker for postganglionic parasympathetic neurons. Using the pre-embedding immunoperoxidase method, we clearly showed that GFP-ir terminals formed synapses predominantly with dendrites and additionally with somata of the ganglion cells. Moreover, bilateral subdiaphragmatic vagotomy caused a marked loss of GFP immunoreactivity in the pancreas. Using a combination of retrograde tracing and immunohistochemistry, we finally demonstrated that nearly half of the pancreas-projecting DMV neurons were immunoreactive for GFP. These results suggest that MC4R-expressing DMV neurons may participate in the regulation of glucose/insulin homeostasis through their projections to the intrapancreatic ganglia.

Glutamatergic neurons in the lateral periaqueductal gray innervate neurokinin-1 receptor-expressing neurons in the ventrolateral medulla of the rat.

The neural pathways underlying the respiratory responses elicited by electrical or chemical stimulation of the lateral part of the periaqueductal gray (lPAG) remain unsettled. In the present study, we examined the lPAG projection to neurokinin-1 receptor (NK1R)-immunoreactive (ir) neurons in the ventrolateral medulla (VLM) which have been implicated in the control of respiration. After biotinylated dextranamine (BDA) injection into the lPAG, NK1R-ir neurons in the rostral VLM were embedded in the plexus of BDA-labeled fibers. At the electron microscopic level, the BDA-labeled terminals made asymmetrical synaptic contacts predominantly with dendrites and additionally with somata of the NK1R-ir neurons. Using retrograde tracing combined with in situ hybridization, we demonstrated that the vast majority of the lPAG neurons projecting to the rostral VLM were positive for vesicular glutamate transporter 2 (VGLUT2) mRNA, but not for glutamic acid decarboxylase 67 mRNA. Using a combination of anterograde tracing and immunohistochemistry, we further demonstrated that the lPAG axon terminals with VGLUT2 immunoreactivity made close apposition with the NK1R-ir neuronal profiles in the rostral VLM. These data suggest that lPAG neurons exert an excitatory influence on NK1R-expressing neurons in the rostral VLM for the control of respiration.

Projections from the anterior basomedial and anterior cortical amygdaloid nuclei to melanin-concentrating hormone-containing neurons in the lateral hypothalamus of the rat.

Melanin-concentrating hormone (MCH) is involved in the regulation of feeding behavior as well as in goal oriented behaviors, and MCH-containing neurons are distributed mainly in the lateral hypothalamic area (LHA). The anterior basomedial nucleus (BMA) and anterior cortical nucleus (CoA) of the amygdala form part of a circuit involved in processing olfactory, gustatory and visceral information, and the BMA-LHA and CoA-LHA pathways are suggested to be implicated in the control of feeding behavior. However, it is still unknown whether or not MCH-containing LHA neurons are under the direct influence of the BMA and CoA. Here the organization of projections from the BMA and CoA to MCH-containing LHA neurons was examined. Using a combined anterograde tracing with biotinylated dextranamine and immunohistochemistry for MCH, we first demonstrated that the distribution pattern of BMA fibers was almost similar to that of CoA fibers in the LHA, and a prominent overlapping distribution of these fibers and MCH-immunoreactive neurons existed in the ventral peripeduncular region of the LHA. We further revealed that asymmetrical synapses were made between these fibers and neurons. Using a combination of retrograde tract-tracing with cholera toxin B subunit and in situ hybridization for vesicular glutamate transporter (VGLUT) 2 mRNA, we finally showed that most of the LHA-projecting BMA and CoA neurons expressed VGLUT2 mRNA. These data suggest that the BMA and CoA of the amygdala may exert excitatory influence upon the MCH-containing LHA neurons for the regulation of feeding behavior.

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.

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.

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.

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.

The key locus of common response inhibition network for no-go and stop signals.

Response inhibition is one of the highest evolved executive functions of human beings. Previous studies revealed a wide variety of brain regions related to response inhibition, although some of them may not be directly related to inhibition but to task-specific effects or noninhibitory cognitive functions such as attention, response competition, or error detection. Here, we conducted event-related functional magnetic resonance imaging studies in which all subjects performed both stop-signal and go/no-go tasks in order to explore key neural correlates within the response inhibition network irrelevant to task designs and other cognitive processes. The successful inhibition in the stop-signal and go/no-go tasks, respectively, activated a set of predominantly right-lateralized hemispheric cortices. The common inhibitory regions across the two tasks included the right middle prefrontal cortex in addition to the right middle occipital cortex. Correlation analysis was carried out within these areas between intensity of activation and behavioral performance in the two tasks. Only the region located in the middle prefrontal cortex showed significant correlations in both tasks. We believe this region is the key locus for execution of response inhibition in the distributed inhibitory neural network.

Glutamatergic neurons in the Kölliker-Fuse nucleus project to the rostral ventral respiratory group and phrenic nucleus: a combined retrograde tracing and in situ hybridization study in the rat.

Kölliker-Fuse nucleus (KF) neurons are considered to excite motoneurons in the phrenic nucleus (PhN) during inspiration through its projection to the PhN and/or to the rostral ventral respiratory group (rVRG), which in turn projects to the PhN, probably by releasing glutamate from their axon terminals. Using a combined retrograde tracing and in situ hybridization technique, here we demonstrate that most of the KF neurons projecting to the PhN and rVRG contain vesicular glutamate transporter 2 (VGLUT2) mRNA but not glutamic acid decarboxylase 67 (GAD67) mRNA, providing definitive evidence that these neurons are glutamatergic. Together with previous data by Stornetta et al. [Stornetta, R.L., Sevigny, C.P., Guyenet, P.G., 2003b. Inspiratory argumenting bulbospinal neurons express both glutamatergic and enkephalinergic phenotypes. J. Comp. Neurol. 455, 113-124], indicating that PhN-projecting rVRG neurons are VGLUT2 mRNA-positive, the present results suggest that the glutamatergic KF-PhN pathway and/or the glutamatergic KF-rVRG-PhN pathway transmit excitatory outputs of KF neurons to the PhN neurons during inspiration.

A light and electron microscopic analysis of the convergent insular cortical and amygdaloid projections to the posterior lateral hypothalamus in the rat, with special reference to cardiovascular function.

The synaptic organization between and among the insular cortex (IC) axons, central amygdaloid nucleus (ACe) axons and posterolateral hypothalamus (PLH) neurons was investigated in the rat using double anterograde tracing and anterograde tracing combined with postembedding immunogold analysis. After ipsilateral injections of biotinylated dextran amine (BDA) into the IC and Phaseolus vulgaris-leucoagglutinin (PHA-L) into the ACe, the conspicuous overlapping distribution of BDA-labeled axon terminals and PHA-L-labeled axon terminals was found in the PLH region just medial to the subthalamic nucleus ipsilateral to the injection sites. At the electron microscopic level, approximately two-thirds of the IC terminals made synapses with small-sized dendrites and the rest did with dendritic spines of the PLH neurons, whereas about 79%, 16% and 5% of the ACe terminals established synapses with small- to medium-sized dendrites, somata, and dendritic spines, respectively, of the PLH neurons. In addition, the IC axon terminals contained densely packed round clear vesicles and their synapses were of asymmetrical type. On the other hand, most of the ACe terminals contained not only pleomorphic clear vesicles but also dense-cored vesicles and their synapses were of symmetrical type although some ACe terminals contained densely packed round clear vesicles and formed asymmetrical synapses. Most of the postsynaptic elements received synaptic inputs from the IC or ACe terminals, and some of single postsynaptic elements received convergent synaptic inputs from both sets of terminals. Furthermore, almost all the ACe terminals were revealed to be immunoreactive for gamma-aminobutyric acid (GABA), by using the anterograde BDA tracing technique combined with immunohistochemistry for GABA. The present data suggest that single PLH neurons are under the excitatory influence of the IC and/or inhibitory influence of the ACe in the circuitry involved in the regulation of cardiovascular functions.

Insular cortical and amygdaloid fibers are in contact with posterolateral hypothalamic neurons projecting to the nucleus of the solitary tract in the rat.

After ipsilateral injections of cholera toxin B subunit (CTb) into the nucleus of the solitary tract (NST) and biotinylated dextran amine (BDA) into the insular cortex (IC) or into the central amygdaloid nucleus (ACe) in the rat, the prominent overlapping distribution of CTb-labeled neurons and BDA-labeled axon terminals was found in the posterolateral hypothalamus (PLH) region just medial to the subthalamic nucleus ipsilateral to the injection sites. At the electron microscopic level, the IC terminals formed asymmetrical synaptic contacts with dendrites and dendritic spines of the NST-projecting PLH neurons, whereas the ACe terminals formed symmetrical synaptic contacts with somata and dendrites of the NST-projecting PLH neurons. The present data suggest that output signals from the IC and ACe may exert excitatory and inhibitory influences, respectively, upon the PLH neurons that project to the NST for regulating cardiovascular functions.

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

After ipsilateral injections of biotinylated dextran amine (BDA) into the central nucleus of the amygdala (ACe) and cholera toxin B subunit (CTb) into the motor trigeminal nucleus (Vm) in the rat, numerous BDA-labeled axons with bouton-like varicosities were distributed bilaterally with a clear-cut ipsilateral dominance in the parvicellular reticular formation (RFp), where many CTb-labeled neurons existed bilaterally with slightly ipsilateral dominance. The prominent overlapping distribution of these labeled axons and neurons was found in the RFp region just ventral to the nucleus of the solitary tract and medial to the spinal trigeminal nucleus throughout the caudalmost part of the pons and the rostral half of the medulla oblongata. Within the neuropil of the RFp region in the rostral half of the medulla oblongata, BDA-labeled axons made symmetrical synaptic contacts predominantly with the dendrites and additionally with the somata of RFp neurons, some of which were labeled with CTb. These data suggest that output signals from the ACe may be transmitted disynaptically to the Vm via the RFp neurons in the medulla oblongata for the control of jaw movements.