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Eupnea is generated by neural circuits located in the ponto-medullary brainstem, but can be modulated by higher brain inputs which contribute to volitional control of breathing and the expression of orofacial behaviors, such as vocalization, sniffing, coughing, and swallowing. Surprisingly, the anatomical organization of descending inputs that connect the forebrain with the brainstem respiratory network remains poorly defined. We hypothesized that descending forebrain projections target multiple distributed respiratory control nuclei across the neuroaxis. To test our hypothesis, we made discrete unilateral microinjections of the retrograde tracer cholera toxin subunit B in the midbrain periaqueductal gray (PAG), the pontine Kölliker-Fuse nucleus (KFn), the medullary Bötzinger complex (BötC), pre-BötC, or caudal midline raphé nuclei. We quantified the regional distribution of retrogradely labeled neurons in the forebrain 12-14 days postinjection. Overall, our data reveal that descending inputs from cortical areas predominantly target the PAG and KFn. Differential forebrain regions innervating the PAG (prefrontal, cingulate cortices, and lateral septum) and KFn (rhinal, piriform, and somatosensory cortices) imply that volitional motor commands for vocalization are specifically relayed via the PAG, while the KFn may receive commands to coordinate breathing with other orofacial behaviors (e.g., sniffing, swallowing). Additionally, we observed that the limbic or autonomic (interoceptive) systems are connected to broadly distributed downstream bulbar respiratory networks. Collectively, these data provide a neural substrate to explain how volitional, state-dependent, and emotional modulation of breathing is regulated by the forebrain., (© 2020 Wiley Periodicals LLC.)

Methadone is an opioid that often leads to fatalities. Interpretation of toxicological findings can be challenging if no further information about the case history is available. The aims of this study were (1) to determine whether brain/blood ratios can assist in the interpretation of methadone findings in fatalities; (2) to examine whether polymorphisms in the gene encoding the P-glycoprotein (also known as multidrug resistance protein 1 (MDR1) or ATP-binding cassette sub-family B member 1 (ABCB1)), which functions as a multispecific efflux pump in the blood-brain barrier, affect brain/blood ratios of methadone. Femoral venous blood and brain tissue (medulla oblongata and cerebellum) from 107 methadone-related deaths were analysed for methadone by gas chromatography-mass spectrometry. In addition, all the samples were genotyped for three common ABCB1 single nucleotide polymorphisms (SNPs rs1045642, rs1128503, and rs2032582) using ion-pair reversed-phase high-performance liquid chromatography-electrospray ionization mass spectrometry (ICEMS). In nearly all cases, methadone concentrations were higher in the brain than in the blood. Inter-individual brain/blood ratios varied (0.6-11.6); the mean ratio was 2.85 (standard deviation 1.83, median 2.35). Moreover, significant differences in mean brain/blood ratios were detected among the synonymous genotypes of rs1045642 in ABCB1 (p = 0.001). Cases with the T/T genotype had significantly higher brain/blood ratios than cases with the other genotypes (T/T vs. T/C difference (d) = 1.54, 95% CI [1.14, 2.05], p = 0.002; T/T vs. C/C d = 1.60, 95% CI [1.13, 2.29], p = 0.004). Our results suggest that the rs1045642 polymorphisms in ABCB1 may affect methadone concentrations in the brain and its site of action and may be an additional factor influencing methadone toxicity.

Columnar structure is a basic unit of the brain, but the mechanism underlying its development remains largely unknown. The medulla, the largest ganglion of the Drosophila melanogaster visual center, provides a unique opportunity to reveal the mechanisms of 3D organization of the columns. In this study, using N-cadherin (Ncad) as a marker, we reveal the donut-like columnar structures along the 2D layer in the larval medulla that evolves to form three distinct layers in pupal development. Column formation is initiated by three core neurons, R8, R7, and Mi1, which establish distinct concentric domains within a column. We demonstrate that Ncad-dependent relative adhesiveness of the core columnar neurons regulates their relative location within a column along a 2D layer in the larval medulla according to the differential adhesion hypothesis. We also propose the presence of mutual interactions among the three layers during formation of the 3D structures of the medulla columns. SIGNIFICANCE STATEMENT The columnar structure is a basic unit of the brain, but its developmental mechanism remains unknown. The medulla, the largest ganglion of the fly visual center, provides a unique opportunity to reveal the mechanisms of 3D organization of the columns. We reveal that column formation is initiated by three core neurons that establish distinct concentric domains within a column. We demonstrate the in vivo evidence of N-cadherin-dependent differential adhesion among the core columnar neurons within a column along a 2D layer in the larval medulla. The 2D larval columns evolve to form three distinct layers in the pupal medulla. We propose the presence of mutual interactions among the three layers during formation of the 3D structures of the medulla columns., (Copyright © 2019 the authors.)

Medullary dorsal horn (MDH), the homolog of spinal dorsal horn, plays essential roles in processing of nociceptive signals from orofacial region toward higher centers, such as the ventral posteromedial thalamic nucleus (VPM) and parafascicular thalamic nucleus (Pf), which belong to the sensory-discriminative and affective aspects of pain transmission systems at the thalamic level, respectively. In the present study, in order to provide morphological evidence for whether neurons in the MDH send collateral projections to the VPM and Pf, a retrograde double tracing method combined with immunofluorescence staining for substance P (SP), SP receptor (SPR) and Fos protein was used. Fluoro-gold (FG) was injected into the VPM and the tetramethylrhodamine-dextran (TMR) was injected into the Pf. The result revealed that both FG- and TMR-labeled projection neurons were observed throughout the entire extent of the MDH, while the FG/TMR double-labeled neurons were mainly located in laminae I and III. It was also found that some of the FG/TMR double-labeled neurons within lamina I expressed SPR and were in close contact with SP-immunoreactive (SP-ir) terminals. After formalin injection into the orofacial region, 41.4% and 34.3% of the FG/TMR double-labeled neurons expressed Fos protein in laminae I and III, respectively. The present results provided morphological evidence for that some SPR-expressing neurons within the MDH send collateral projections to both VPM and Pf and might be involved in sensory-discriminative and affective aspects of acute orofacial nociceptive information transmission., (Copyright © 2019 IBRO. Published by Elsevier Ltd. All rights reserved.)

Aim: To obtain the data on the spatial relationships between catecholamine (TH-positive) and nitroxidergic (nNOS-positive) neurons in vasomotor nuclei of the medulla in different periods of hypertension development., Material and Methods: The experiment was performed on male Wistar rats (n=45) with induced renovascular hypertension (RVH). TH and nNOS in neurons of solitary tract nuclei, reticular small-and giant cell nuclei were detected using immunohistochemical methods., Results and Conclusion: The most early and severe changes in the intensity of reaction and amount of nNOS-positive neurons were noted in the solitary tract nucleus. Significant changes in the quantitative parameters of TH-positive neurons in RVH were identified only in the reticular giant cell nucleus but they appeared later and were less expressed compared to nNOS-positive cells. This resulted in the changes of spatial relationships between two types of neurons and remodeling of the bulbar region of the cardiovascular center.

The retrotrapezoid nucleus (RTN) consists, by definition, of Phox2b-expressing, glutamatergic, non-catecholaminergic, noncholinergic neurons located in the parafacial region of the medulla oblongata. An unknown proportion of RTN neurons are central respiratory chemoreceptors and there is mounting evidence for biochemical diversity among these cells. Here, we used multiplexed in situ hybridization and single-cell RNA-Seq in male and female mice to provide a more comprehensive view of the phenotypic diversity of RTN neurons. We now demonstrate that the RTN of mice can be identified with a single and specific marker, Neuromedin B mRNA ( Nmb ). Most (∼75%) RTN neurons express low-to-moderate levels of Nmb and display chemoreceptor properties. Namely they are activated by hypercapnia, but not by hypoxia, and express proton sensors, TASK-2 and Gpr4. These Nmb -low RTN neurons also express varying levels of transcripts for Gal , Penk , and Adcyap1 , and receptors for substance P, orexin, serotonin, and ATP. A subset of RTN neurons (∼20-25%), typically larger than average, express very high levels of Nmb mRNA. These Nmb -high RTN neurons do not express Fos after hypercapnia and have low-to-undetectable levels of Kcnk5 or Gpr4 transcripts; they also express Adcyap1 , but are essentially devoid of Penk and Gal transcripts. In male rats, Nmb is also a marker of the RTN but, unlike in mice, this gene is expressed by other types of nearby neurons located within the ventromedial medulla. In sum, Nmb is a selective marker of the RTN in rodents; Nmb -low neurons, the vast majority, are central respiratory chemoreceptors, whereas Nmb -high neurons likely have other functions. SIGNIFICANCE STATEMENT Central respiratory chemoreceptors regulate arterial PCO 2 by adjusting lung ventilation. Such cells have recently been identified within the retrotrapezoid nucleus (RTN), a brainstem nucleus defined by genetic lineage and a cumbersome combination of markers. Using single-cell RNA-Seq and multiplexed in situ hybridization, we show here that a single marker, Neuromedin B mRNA ( Nmb ), identifies RTN neurons in rodents. We also suggest that >75% of these Nmb neurons are chemoreceptors because they are strongly activated by hypercapnia and express high levels of proton sensors ( Kcnk5 and Gpr4 ). The other RTN neurons express very high levels of Nmb , but low levels of Kcnk5/Gpr4/pre-pro-galanin/pre-pro-enkephalin , and do not respond to hypercapnia. Their function is unknown., (Copyright © 2017 the authors 0270-6474/17/3711744-14$15.00/0.)

Nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) enzymatic activity has been reported in few amphibian species. In this study, we report its unusual localization in the medulla oblongata, spinal cord, cranial nerves, spinal nerves, and ganglions of the frog, Microhyla ornata. In the rhombencephalon, at the level of facial and vagus nerves, the NADPH-d labeling was noted in the nucleus of the abducent and facial nerves, dorsal nucleus of the vestibulocochlear nerve, the nucleus of hypoglossus nerve, dorsal and lateral column nucleus, the nucleus of the solitary tract, the dorsal field of spinal grey, the lateral and medial motor fields of spinal grey and radix ventralis and dorsalis (2-10). Many ependymal cells around the lining of the fourth ventricle, both facial and vagus nerves and dorsal root ganglion, were intensely labeled with NADPH-d. Most strikingly the NADPH-d activity was seen in small and large sized motoneurons in both medial and lateral motor neuron columns on the right and left sides of the brain. This is the largest stained group observed from the caudal rhombencephalon up to the level of radix dorsalis 10 in the spinal cord. The neurons were either oval or elongated in shape with long processes and showed significant variation in the nuclear and cellular diameter. A massive NADPH-d activity in the medulla oblongata, spinal cord, and spinal nerves implied an important role of this enzyme in the neuronal signaling as well as in the modulation of motor functions in the peripheral nervous systems of the amphibians., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Previously, we demonstrated that chronic exposure to low levels of estradiol-17β (E2) increases mean arterial pressure (MAP) in young female Sprague-Dawley (SD) rats, however, the underlying mechanisms are unclear. Since endothelin-1 (ET-1) is implicated in blood pressure (BP) regulation, we hypothesized that E2's effects on MAP are mediated through central ET-1. To test this, young female SD rats were either sham implanted or implanted s.c. with slow-release E2 pellets (20 ng/day for 90 days). BP was monitored by telemetry. After 75 days of E2 exposure, ET A antagonist or vehicle was administered i.c.v. After 90 days of E2 exposure, rats were sacrificed, and the paraventricular nucleus (PVN) and rostral ventrolateral medulla (RVLM) were microdissected for gene expression and protein analysis of ET-1 and its receptors. E2 exposure increased MAP after pellet implantation. Gene expression of ET-1 and ET A but not ET B receptors were upregulated in the PVN and RVLM of E2 treated animals. Further, the protein levels of ET A receptor were also increased in the PVN of E2 treated animals. However, i.c.v. infusion of the ET A antagonist did not completely block the increase in blood pressure. Our results suggest that increases in central ET-1 activity could possibly play a role in chronic E2-induced increase in BP but further studies are needed to completely understand the contribution of ET-1 in this phenomenon.

It has been suggested that the trigemino-thalamic and trigemino-parabrachial projection neurons in the medullary dorsal horn (MDH) are highly implicated in the sensory-discriminative and emotional/affective aspects of orofacial pain, respectively. In previous studies, some neurons were reported to send projections to both the thalamus and parabrachial nucleus by way of collaterals in the MDH. However, little is known about the chemoarchitecture of this group of neurons. Thus, in the present study, we determined whether the neurokinin-1 (NK-1) receptor, which is crucial for primary orofacial pain signaling, was expressed in MDH neurons co-innervating the thalamus and parabrachial nucleus. Vesicular glutamate transporter 2 (VGLUT2) mRNA, a biomarker for the subgroup of glutamatergic neurons closely related to pain sensation, was assessed in trigemino-parabrachial projection neurons in the MDH. After stereotactic injection of fluorogold (FG) and cholera toxin subunit B (CTB) into the ventral posteromedial thalamic nucleus (VPM) and parabrachial nucleus (PBN), respectively, triple labeling with fluorescence dyes for FG, CTB and NK-1 receptor (NK-1R) revealed that approximately 76 % of the total FG/CTB dually labeled neurons were detected as NK-1R-immunopositive, and more than 94 % of the triple-labeled neurons were distributed in lamina I. In addition, by FG retrograde tract-tracing combined with fluorescence in situ hybridization (FISH) for VGLUT2 mRNA, 54, 48 and 70 % of FG-labeled neurons in laminae I, II and III, respectively, of the MDH co-expressed FG and VGLUT2 mRNA. Thus, most of the MDH neurons co-innervating the thalamus and PBN were glutamatergic. Most MDH neurons providing the collateral axons to both the thalamus and parabrachial nucleus in rats were NK-1R-immunopositive and expressed VGLUT2 mRNA. NK-1R and VGLUT2 in MDH neurons may be involved in both sensory-discriminative and emotional/affective aspects of orofacial pain processing.

Evidence has suggested that cerebrospinal fluid-contacting nucleus (CSF-contacting nucleus) is correlated with the development and recurrence of pain. A recent research showed that the CSF-contacting nucleus acts as a component of the descending 5-hydroxytryptamine (5-HT) system and plays a role in descending pain inhibition. However, limited studies are conducted to investigate the relationship between the CSF-contacting nucleus and pain. In present study, we explored the effect of CSF-contacting nucleus on nociceptive behaviors in both normal and neuropathic rats via targeted ablation of the CSF-contacting nucleus in the brainstem, using cholera toxin subunit B-saporin (CB-SAP), a cytotoxin coupled to cholera toxin subunit B. The CB-SAP-treated rats showed aggravated thermal hyperalgesia and mechanical allodynia. Also, results from immunohistochemical experiments showed that rostral ventromedial medulla (RVM) received fiber projection from the CSF-contacting nucleus, which disappeared after ablation of the CSF-contacting nucleus, and the CB-SAP treated rats showed downregulation of c-Fos expression in the RVM as compared with the rats receiving i.c.v. injection of phosphate buffer saline (PBS). A significant downregulation of 5-HT-labeled neurons and tryptophan hydroxylase 2 (TPH2) as the marker of 5-HT cells in the RVM, and 5-HT expression in spinal dorsal horn in both normal and chronic constriction injury (CCI) rats after i.c.v. injection of CB-SAP was observed. These results suggested that RVM may be involved in descending pain modulation originating from the CSF-contacting nucleus.

In the dorsal facial area (DFA) of the medulla, an activation of either P2 purinergic receptor or nitric oxide synthase (NOS) results in the release of glutamate, leading to an increase in blood flow of the common carotid artery (CCA). It is not known whether activation of the P2 receptor by ATP may mediate activation of NOS/guanylyl cyclase to cause glutamate release and/or whether L-Arg (nitric oxide (NO) precursor) may also cause ATP release from any other neuron, to cause an increase in CCA flow. We demonstrated that microinjections of P2 receptor agonists (ATP, α,β-methylene ATP) or NO precursor (L-arginine) into the DFA increased CCA blood flow. The P2-induced CCA blood flow increase was dose-dependently reduced by pretreatment with NG-nitro-arginine methyl ester (L-NAME, a non-specific NOS inhibitor), 7-nitroindazole (7-NI, a relatively selective neuronal NOS inhibitor) or methylene blue (MB, a guanylyl cyclase inhibitor) but not by that with D-NAME (an isomer of L-NAME) or N5-(1-iminoethyl)-L-ornithine (L-NIO, a potent endothelial NOS inhibitor). Involvement of glutamate release in these responses were substantiated by microdialysis studies, in which perfusions of ATP into the DFA increased the glutamate concentration in dialysates, but co-perfusion of ATP with L-NAME or 7-NI did not. Nevertheless, the arginine-induced CCA blood flow increase was abolished by combined pretreatment of L-NAME and MB, but not affected by pretreatment with a selective P2 receptor antagonist, pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid (PPADS). In conclusion, ATP activation of the P2 receptor in the DFA induced activation of neuronal NOS/guanylyl cyclase, which causes glutamate release leading to an increase in CCA blood flow. However, arginine activation of neuronal NOS/guanylyl cyclase, which also caused glutamate release and CCA blood flow increase, did not induce activation of P2 receptors. These findings provide important information for drug design and/or developing therapeutic strategies for the diseases associated with CCA blood flow that supplies intra- and extra-cranial tissues., (Copyright © 2014 IBRO. Published by Elsevier Ltd. All rights reserved.)