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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., (© 2013 Elsevier B.V. All rights reserved.)

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., (Copyright © 2012 Elsevier Ireland Ltd and the Japan Neuroscience Society. All rights reserved.)

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 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.

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.

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.

The hippocampal formation and amygdala are responsible for regulating emotion, learning, and behavior. The hippocampal projection to the amygdala has been demonstrated to originate in the subiculum and adjacent portion of field CA1 of the Ammon's horn (Sub/CA1) in the rat; however, the topographical organization of this pathway is still understudied. To make it clear, we performed anterograde and retrograde tracing with biotinylated dextran amine (BDA) and cholera toxin B subunit (CTb), respectively, in the rat. A series of BDA experiments revealed that the temporal-to-septal axis of origin determined a medial-to-lateral axis of termination in the amygdala. Briefly, the temporal region of the Sub/CA1 projects preferentially to the medial amygdaloid region including the medial, intercalated, and basomedial nuclei and the amygdalohippocampal transition area, and progressively more septal portions of the Sub/CA1 distribute their efferents in more lateral regions of the amygdala. Sub/CA1 fibers distributed in the central amygdaloid nucleus were relatively few. Retrograde tracing with CTb confirmed this topography and revealed little hippocampal innervation of the central nucleus of the amygdala. These observations suggest that distinct Sub/CA1 regions arranged along the longitudinal hippocampal axis may influence distinct modalities of the amygdala function., (Copyright 2006 Wiley-Liss, Inc.)

After ipsilateral injections of biotinylated dextran amine (BDA) into the Kölliker-Fuse (KF) nucleus and cholera toxin B subunit (CTb) into the ventral horn in C4 to C5 segments of the spinal cord, an overlapping distribution of BDA-labeled axon terminals and CTb-labeled neurons was found in the rostral ventral respiratory group (rVRG) region ipsilateral to the injection sites. After ipsilateral injections of BDA into the KF and Fluoro-Gold (FG) into the ventral horn in C4 to C5 segments of the spinal cord, BDA-labeled axons were found to make asymmetrical synapses with the somata and dendrites of FG-labeled neurons within the neuropil of the rVRG region. Using retrograde tracing combined with immunohistochemistry for phosphate-activated glutaminase (PAG), we observed that as many as 72% of the rVRG neurons projecting to the PhN were immunoreactive for PAG and that approximately 62% and 75% of the KF neurons projecting respectively to the rVRG region and PhN contain PAG immunoreactivity. Using anterograde tracing combined with immunohistochemistry for vesicular glutamate transporter 2 (VGluT2), we further demonstrated that the KF axon terminals in the rVRG and PhN regions as well as the rVRG axon terminals in the PhN region contain VGluT2 immunoreactivity. The present results suggest that the glutamatergic pathways from the KF to the PhN directly and indirectly via the rVRG region may exist and underlie the inspiratory responses that are elicited by activation of the KF neurons.

The present tract-tracing study in the rat indicated that neurons in the ventrolateral part of the parafascicular thalamic nucleus (PF), where nigral fibers from the dorsolateral part of the substantia nigra pars reticulata (SNr) terminated, sent their axons to the ventrolateral part of the striatum as well as to the rostrolateral part of the lateral agranular cortex (AGl). We further demonstrated that symmetrical synaptic contacts were made between these nigral axons and striatum- or AGl-projecting PF neurons. Since the dorsolateral part of the SNr, ventrolateral part of the striatum and rostrolateral part of the AGl are responsible regions for orofacial behaviors, the nigrothalamostriatal and nigrothalamo-cortical pathways via the ventrolateral part of the PF may play a role in the control of orofacial motor function., (Copyright 2003 Lippincott Williams & Wilkins)

The ventrolateral part of the parafascicular thalamic nucleus (PF), which is considered to take part in the control mechanism of orofacial motor functions, receives projection fibers not only from the dorsolateral part of the substantia nigra pars reticulata (SNr) but also from the ventral part of the reticular thalamic nucleus (RT) [Tsumori et al., Brain Res. 858 (2000) 429]. In order to better understand the influence of these fibers upon the PF projection neurons, the morphology, synaptology and chemical nature of them were examined in the present study. After ipsilateral injections of Phaseolus vulgaris-leucoagglutinin (PHA-L) into the dorsolateral part of the SNr and biotinylated dextran amine (BDA) into the ventral part of the RT, overlapping distributions of PHA-L-labeled SNr fibers and BDA-labeled RT fibers were seen in the ventrolateral part of the PF. At the electron microscopic level, the SNr terminals made synapses predominantly with the medium to small dendrites and far less frequently with the somata and large dendrites, whereas approximately half of the RT terminals made synapses with the somata and large dendrites and the rest did with the medium to small dendrites of PF neurons. Some of single dendritic as well as single somatic profiles received convergent synaptic inputs from both sets of terminals. These terminals were packed with pleomorphic synaptic vesicles and formed symmetrical synapses. After combined injections of PHA-L into the dorsolateral part of the SNr, BDA into the ventral part of the RT and wheat germ agglutinin-horseradish peroxidase (WGA-HRP) into the ventrolateral part of the striatum or into the rostroventral part of the lateral agranular cortex, WGA-HRP-labeled neurons were embedded in the plexus of PHA-L- and BDA-labeled axon terminals within the ventrolateral part of the PF, where the PHA-L- and/or BDA-labeled terminals were in synaptic contact with single somatic and dendritic profiles of the WGA-HRP-labeled neurons. Furthermore, the SNr and RT axon 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 GABAergic SNr and RT fibers may exert different inhibitory influences on the PF neurons for regulating the thalamic outflow from the PF to the cerebral cortex and/or striatum in the control of orofacial movements.

The parabrachial complex, which plays an important role in respiratory regulation, has been reported to send projection fibers to the phrenic nucleus, but the synaptic organization between the parabrachial fibers and the phrenic motoneurons has not been examined. Using anterograde and retrograde tracing methods, we found in the rat that the parabrachial fibers originating mainly from the Kölliker-Fuse nucleus (KF) terminated not only within the phrenic nucleus but also on the radial dendritic bundles of the phrenic motoneurons. It was further revealed that the KF fibers made asymmetrical synapses predominantly with dendrites and partly with somata of the phrenic motoneurons. These data suggest that output signals from the KF may exert excitatory influence directly upon the phrenic motoneurons.

We observed by anterograde and retrograde tracing techniques that projection fibers originating from the medial vestibular nucleus (MVe) of the rat terminated in the dorsal two-thirds of the lateral part of the parafascicular thalamic nucleus (PF), where neurons sending their axons to the dorsolateral part of the striatum existed. It was further revealed that the vestibular fibers made asymmetrical synaptic contacts mainly with dendrites and additionally with soma of the striatum-projecting PF neurons. These data suggest that output signals from the MVe may be transmitted disynaptically to the striatal neurons via the PF neurons.

It was revealed in the rat that single neurons in the substantia nigra pars reticulata (SNr) innervated both the superior colliculus (SC) and the parvicellular reticular formation (RFp) in the pons and medulla oblongata by way of axon collaterals. After injecting Fluoro-gold into the lateral part of the SC and Fluoro-ruby into the RFp on the same side, some SNr neurons were double-labeled with both tracers. They were localized in the dorsolateral part of the caudal half of the SNr ipsilateral to the injection sites.