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Acid sensitivity and ultrastructure of the retrotrapezoid nucleus in Phox2b-EGFP transgenic mice.

ABSTRACT: The retrotrapezoid nucleus (RTN) contains noncholinergic noncatecholaminergic glutamatergic neurons that express the transcription factor Phox2b (chemically coded or "cc" RTN neurons). These cells regulate breathing and may be central chemoreceptors. Here we explore their ultrastructure and their acid sensitivity by using two novel BAC eGFP-Phox2b transgenic mice (B/G, GENSAT JX99) in which, respectively, 36% and 100% of the cc RTN neurons express the transgene in complete or partial anatomical isolation from other populations of eGFP neurons. All but one of the eGFP-labeled RTN neurons recorded in these mice were acid activated in slices. These cells contained VGLUT2 mRNA, and 50% contained preprogalanin mRNA (determined by single-cell PCR in the B/G mouse). Two neuronal subgroups were revealed, which differed in discharge rate at pH 7.3 (type I approximately 2; type II approximately 4 Hz) and the degree of alkalization that silenced the cells (type I 7.4-7.6, type II 7.8-8.0). Medial to the RTN, C1 neurons recorded in a tyrosine hydroxylase-GFP mouse were pH insensitive between pH 6.9 and pH 7.5. Ultrastructural studies demonstrated that eGFP-labeled RTN neurons were surrounded by numerous capillaries and were often in direct contact with glial cells, pericytes, and the basement membrane of capillaries. Terminals contacting large proximal eGFP dendrites formed mainly symmetric, likely inhibitory, synapses. Terminals on more distal eGFP dendrites formed preferentially asymmetric, presumably excitatory, synapses. In sum, C1 cells are pH insensitive, whereas cc RTN neurons are uniformly acid sensitive. The RTN neurons receive inhibitory and excitatory synaptic inputs and may have unfettered biochemical interactions with glial cells and the local microvasculature.

SUBMITTER: Lazarenko RM

PROVIDER: S-EPMC2826801 | biostudies-literature | 2009 Nov

REPOSITORIES: biostudies-literature

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Acid sensitivity and ultrastructure of the retrotrapezoid nucleus in Phox2b-EGFP transgenic mice.

Lazarenko Roman M RM Milner Teresa A TA Depuy Seth D SD Stornetta Ruth L RL West Gavin H GH Kievits Justin A JA Bayliss Douglas A DA Guyenet Patrice G PG

The Journal of comparative neurology 20091101 1

The retrotrapezoid nucleus (RTN) contains noncholinergic noncatecholaminergic glutamatergic neurons that express the transcription factor Phox2b (chemically coded or "cc" RTN neurons). These cells regulate breathing and may be central chemoreceptors. Here we explore their ultrastructure and their acid sensitivity by using two novel BAC eGFP-Phox2b transgenic mice (B/G, GENSAT JX99) in which, respectively, 36% and 100% of the cc RTN neurons express the transgene in complete or partial anatomical ...[more]

PMID: 19711410

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Project description: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 PCO2 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.

Project description:BackgroundVolatile anesthetics moderately depress respiratory function at clinically relevant concentrations. Phox2b-expressing chemosensitive neurons in the retrotrapezoid nucleus, a respiratory control center, are activated by isoflurane, but the underlying mechanisms remain unclear. The hypothesis of this study was that the sodium leak channel contributes to the volatile anesthetics-induced modulation of retrotrapezoid nucleus neurons and to respiratory output.MethodsThe contribution of sodium leak channels to isoflurane-, sevoflurane-, and propofol-evoked activity of Phox2b-expressing retrotrapezoid nucleus neurons and respiratory output were evaluated in wild-type and genetically modified mice lacking sodium leak channels (both sexes). Patch-clamp recordings were performed in acute brain slices. Whole-body plethysmography was used to measure the respiratory activity.ResultsIsoflurane at 0.42 to 0.50 mM (~1.5 minimum alveolar concentration) increased the sodium leak channel-mediated holding currents and conductance from -75.0 ± 12.9 to -130.1 ± 34.9 pA (mean ± SD, P = 0.002, n = 6) and 1.8 ± 0.5 to 3.6 ± 1.0 nS (P = 0.001, n = 6), respectively. At these concentrations, isoflurane increased activity of Phox2b-expressing retrotrapezoid nucleus neurons from 1.1 ± 0.2 to 2.8 ± 0.2 Hz (P < 0.001, n = 5), which was eliminated by bath application of gadolinium or genetic silencing of sodium leak channel. Genetic silencing of sodium leak channel in the retrotrapezoid nucleus resulted in a diminished ventilatory response to carbon dioxide in mice under control conditions and during isoflurane anesthesia. Sevoflurane produced an effect comparable to that of isoflurane, whereas propofol did not activate sodium leak channel-mediated holding conductance.ConclusionsIsoflurane and sevoflurane increase neuronal excitability of chemosensitive retrotrapezoid nucleus neurons partly by enhancing sodium leak channel conductance. Sodium leak channel expression in the retrotrapezoid nucleus is required for the ventilatory response to carbon dioxide during anesthesia by isoflurane and sevoflurane, thus identifying sodium leak channel as a requisite determinant of respiratory output during anesthesia of volatile anesthetics.Editor’s perspective

Project description:Chronic migraine (CM) is a highly disabling neurological disorder characterized by recurrent headache accompanied by a variety of sensory and/or emotional symptoms. However, the mechanisms of migraine onset and its chronicity have not been elucidated. The present study was designed to search for brain regions and neurons that were abnormally activated by CM and might be related to its pathogenesis and different concomitant symptoms. CM models were established here by repeated intraperitoneal injection of nitroglycerin (NTG) every other day for 9 days to early growth response gene 1 (Egr1)-enhanced green fluorescent protein (EGFP) transgenic mice, which allowed monitoring of neuronal activities in the whole brain. CM-related behaviors were recorded through head grooming test and light aversion assay. Elevation of Egr1 expression signals was detected in trigeminal nucleus caudalis (TNC), primary somatosensory cortex (SSp), lateral amygdala nucleus (LA), primary visual area (VISp), and temporal association areas (TEa) 2 h after the last injection of NTG by immunofluorescence and digital slice scanning technology. Meanwhile, no change of Egr1 expression was found in auditory areas (AUD), CA1, ectorhinal area (ECT), piriform (PIR), and anterior cingulate area (ACC). Furthermore, with the strongest support by evidence-based medicine among the current limited oral treatments of CM, topiramate was administrated every day for 11 days from 2 days before the first NTG injection. The results showed that topiramate partially improved the photophobia behavior of CM models in the short-term with gradually weakened efficacy as the course of the disease prolonged. Meanwhile, NTG-induced increase in Egr1 expression was completely reversed in TNC, SSp, and VISp and partially reduced in LA and TEa by topiramate at the same time point mentioned above. In conclusion, the current results suggested that the abnormal hyperactivities in TNC, SSp and VISp were associated with the pathogenesis of CM.

Dataset Information

Acid sensitivity and ultrastructure of the retrotrapezoid nucleus in Phox2b-EGFP transgenic mice.

Publications

Acid sensitivity and ultrastructure of the retrotrapezoid nucleus in Phox2b-EGFP transgenic mice.

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