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A Phox2b BAC Transgenic Rat Line Useful for Understanding Respiratory Rhythm Generator Neural Circuitry.

Ikeda K, Takahashi M, Sato S, Igarashi H, Ishizuka T, Yawo H, Arata S, Southard-Smith EM, Kawakami K, Onimaru H - PLoS ONE (2015)

Bottom Line: Here we describe the generation of a novel transgenic (Tg) rat harboring fluorescently labeled Pre-I neurons in the RTN/pFRG.In addition, the Tg rat showed fluorescent signals in autonomic enteric neurons and carotid bodies.Because the Tg rat expresses inducible Cre recombinase in PHOX2B-positive cells during development, it is a potentially powerful tool for dissecting the entire picture of the respiratory neural network during development and for identifying the CO2/O2 sensor molecules in the adult central and peripheral nervous systems.

View Article: PubMed Central - PubMed

Affiliation: Division of Biology, Hyogo College of Medicine, Nishinomiya, Hyogo, Japan; Division of Biology, Center for Molecular Medicine, Jichi Medical University, Shimotsuke, Tochigi, Japan.

ABSTRACT
The key role of the respiratory neural center is respiratory rhythm generation to maintain homeostasis through the control of arterial blood pCO2/pH and pO2 levels. The neuronal network responsible for respiratory rhythm generation in neonatal rat resides in the ventral side of the medulla and is composed of two groups; the parafacial respiratory group (pFRG) and the pre-Bötzinger complex group (preBötC). The pFRG partially overlaps in the retrotrapezoid nucleus (RTN), which was originally identified in adult cats and rats. Part of the pre-inspiratory (Pre-I) neurons in the RTN/pFRG serves as central chemoreceptor neurons and the CO2 sensitive Pre-I neurons express homeobox gene Phox2b. Phox2b encodes a transcription factor and is essential for the development of the sensory-motor visceral circuits. Mutations in human PHOX2B cause congenital hypoventilation syndrome, which is characterized by blunted ventilatory response to hypercapnia. Here we describe the generation of a novel transgenic (Tg) rat harboring fluorescently labeled Pre-I neurons in the RTN/pFRG. In addition, the Tg rat showed fluorescent signals in autonomic enteric neurons and carotid bodies. Because the Tg rat expresses inducible Cre recombinase in PHOX2B-positive cells during development, it is a potentially powerful tool for dissecting the entire picture of the respiratory neural network during development and for identifying the CO2/O2 sensor molecules in the adult central and peripheral nervous systems.

No MeSH data available.


Related in: MedlinePlus

Pre-I neurons in Phox2b-EYFP/CreERT2 Tg rats are EYFP-positive.A, Burst pattern of the recorded neuron in the rostral pFRG of newborn Tg rat shows typical Pre-I neuron discharge. MP, membrane potential; C4, fourth cervical ventral root activity. B, Membrane potential response to high CO2 stimulation (2% CO2 → 8% CO2) in the presence of tetrodotoxin (TTX). Negative deflections of the baseline membrane potential are proportional to input resistance, indicating that the neuron is CO2 sensitive. C-F, Coronal sections of brainstem-spinal cord preparation after electrophysiological analysis (A, B) to identify the location of the recorded neuron stained by Texas Red (yellow arrows). The recorded neuron is EYFP-positive and PHOX2B-immunoreactive. G, Distribution of EYFP-positive Pre-I neurons with CO2 sensitivity, plotted in the corresponding slice at the level of 500–600 μm rostral to the caudal end of the facial nucleus. Each dot represents a single recorded neuron (n = 5). nVII, facial nucleus; CST, corticospinal tract.
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pone.0132475.g006: Pre-I neurons in Phox2b-EYFP/CreERT2 Tg rats are EYFP-positive.A, Burst pattern of the recorded neuron in the rostral pFRG of newborn Tg rat shows typical Pre-I neuron discharge. MP, membrane potential; C4, fourth cervical ventral root activity. B, Membrane potential response to high CO2 stimulation (2% CO2 → 8% CO2) in the presence of tetrodotoxin (TTX). Negative deflections of the baseline membrane potential are proportional to input resistance, indicating that the neuron is CO2 sensitive. C-F, Coronal sections of brainstem-spinal cord preparation after electrophysiological analysis (A, B) to identify the location of the recorded neuron stained by Texas Red (yellow arrows). The recorded neuron is EYFP-positive and PHOX2B-immunoreactive. G, Distribution of EYFP-positive Pre-I neurons with CO2 sensitivity, plotted in the corresponding slice at the level of 500–600 μm rostral to the caudal end of the facial nucleus. Each dot represents a single recorded neuron (n = 5). nVII, facial nucleus; CST, corticospinal tract.

Mentions: We reported previously that the majority of Pre-I neurons in the rostral pFRG were PHOX2B positive and that the PHOX2B-positive Pre-I neurons in neonatal rat were glutamatergic and expressed neurokinin 1 receptor (NK1R) [22, 59] (data not shown), consistent with previous reports by other investigators [12, 13, 65]. To investigate whether the EYFP-positive cells in the pFRG of this novel Tg rat indeed show the characteristics of the Pre-I neurons, we performed whole-cell patch clamp recordings from EYFP-positive cells in the region of the rostral pFRG of the neonatal rat (n = 5). The average resting membrane potential was -50.0±2.6 mV with an input resistance of 783±203 MΩ in standard solution (5% CO2) (Fig 5A). The neurons were depolarized in response to hypercapnia (2% → 8%) in the presence of 0.5 μM TTX, accompanied by an increase in input resistance (Fig 6B) (2% CO2: -46.8 ± 4.3 mV, 374 ± 141 MΩ, 8% CO2: -38.8 ± 6.5 mV (P<0.05), 537 ± 237 MΩ (P<0.05), respectively). These results indicate that the mechanism of the neuronal CO2 response was postsynaptic and probably mediated by closing K+ channels, as discussed previously [23, 58]. These neurons were marked by Texas Red during recording. After recording, we confirmed that they were immunoreactive with PHOX2B (Fig 6C–6F). The results indicate that the EYFP-positive cells in the pFRG have the characteristic of Pre-I neurons. We mapped the distribution of CO2-sensitive EYFP-positive Pre-I neurons in five preparations (Fig 6G). The results showed dense localization of these neurons in the superficial area just ventral to the facial nucleus, as described previously [22].


A Phox2b BAC Transgenic Rat Line Useful for Understanding Respiratory Rhythm Generator Neural Circuitry.

Ikeda K, Takahashi M, Sato S, Igarashi H, Ishizuka T, Yawo H, Arata S, Southard-Smith EM, Kawakami K, Onimaru H - PLoS ONE (2015)

Pre-I neurons in Phox2b-EYFP/CreERT2 Tg rats are EYFP-positive.A, Burst pattern of the recorded neuron in the rostral pFRG of newborn Tg rat shows typical Pre-I neuron discharge. MP, membrane potential; C4, fourth cervical ventral root activity. B, Membrane potential response to high CO2 stimulation (2% CO2 → 8% CO2) in the presence of tetrodotoxin (TTX). Negative deflections of the baseline membrane potential are proportional to input resistance, indicating that the neuron is CO2 sensitive. C-F, Coronal sections of brainstem-spinal cord preparation after electrophysiological analysis (A, B) to identify the location of the recorded neuron stained by Texas Red (yellow arrows). The recorded neuron is EYFP-positive and PHOX2B-immunoreactive. G, Distribution of EYFP-positive Pre-I neurons with CO2 sensitivity, plotted in the corresponding slice at the level of 500–600 μm rostral to the caudal end of the facial nucleus. Each dot represents a single recorded neuron (n = 5). nVII, facial nucleus; CST, corticospinal tract.
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Related In: Results  -  Collection

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pone.0132475.g006: Pre-I neurons in Phox2b-EYFP/CreERT2 Tg rats are EYFP-positive.A, Burst pattern of the recorded neuron in the rostral pFRG of newborn Tg rat shows typical Pre-I neuron discharge. MP, membrane potential; C4, fourth cervical ventral root activity. B, Membrane potential response to high CO2 stimulation (2% CO2 → 8% CO2) in the presence of tetrodotoxin (TTX). Negative deflections of the baseline membrane potential are proportional to input resistance, indicating that the neuron is CO2 sensitive. C-F, Coronal sections of brainstem-spinal cord preparation after electrophysiological analysis (A, B) to identify the location of the recorded neuron stained by Texas Red (yellow arrows). The recorded neuron is EYFP-positive and PHOX2B-immunoreactive. G, Distribution of EYFP-positive Pre-I neurons with CO2 sensitivity, plotted in the corresponding slice at the level of 500–600 μm rostral to the caudal end of the facial nucleus. Each dot represents a single recorded neuron (n = 5). nVII, facial nucleus; CST, corticospinal tract.
Mentions: We reported previously that the majority of Pre-I neurons in the rostral pFRG were PHOX2B positive and that the PHOX2B-positive Pre-I neurons in neonatal rat were glutamatergic and expressed neurokinin 1 receptor (NK1R) [22, 59] (data not shown), consistent with previous reports by other investigators [12, 13, 65]. To investigate whether the EYFP-positive cells in the pFRG of this novel Tg rat indeed show the characteristics of the Pre-I neurons, we performed whole-cell patch clamp recordings from EYFP-positive cells in the region of the rostral pFRG of the neonatal rat (n = 5). The average resting membrane potential was -50.0±2.6 mV with an input resistance of 783±203 MΩ in standard solution (5% CO2) (Fig 5A). The neurons were depolarized in response to hypercapnia (2% → 8%) in the presence of 0.5 μM TTX, accompanied by an increase in input resistance (Fig 6B) (2% CO2: -46.8 ± 4.3 mV, 374 ± 141 MΩ, 8% CO2: -38.8 ± 6.5 mV (P<0.05), 537 ± 237 MΩ (P<0.05), respectively). These results indicate that the mechanism of the neuronal CO2 response was postsynaptic and probably mediated by closing K+ channels, as discussed previously [23, 58]. These neurons were marked by Texas Red during recording. After recording, we confirmed that they were immunoreactive with PHOX2B (Fig 6C–6F). The results indicate that the EYFP-positive cells in the pFRG have the characteristic of Pre-I neurons. We mapped the distribution of CO2-sensitive EYFP-positive Pre-I neurons in five preparations (Fig 6G). The results showed dense localization of these neurons in the superficial area just ventral to the facial nucleus, as described previously [22].

Bottom Line: Here we describe the generation of a novel transgenic (Tg) rat harboring fluorescently labeled Pre-I neurons in the RTN/pFRG.In addition, the Tg rat showed fluorescent signals in autonomic enteric neurons and carotid bodies.Because the Tg rat expresses inducible Cre recombinase in PHOX2B-positive cells during development, it is a potentially powerful tool for dissecting the entire picture of the respiratory neural network during development and for identifying the CO2/O2 sensor molecules in the adult central and peripheral nervous systems.

View Article: PubMed Central - PubMed

Affiliation: Division of Biology, Hyogo College of Medicine, Nishinomiya, Hyogo, Japan; Division of Biology, Center for Molecular Medicine, Jichi Medical University, Shimotsuke, Tochigi, Japan.

ABSTRACT
The key role of the respiratory neural center is respiratory rhythm generation to maintain homeostasis through the control of arterial blood pCO2/pH and pO2 levels. The neuronal network responsible for respiratory rhythm generation in neonatal rat resides in the ventral side of the medulla and is composed of two groups; the parafacial respiratory group (pFRG) and the pre-Bötzinger complex group (preBötC). The pFRG partially overlaps in the retrotrapezoid nucleus (RTN), which was originally identified in adult cats and rats. Part of the pre-inspiratory (Pre-I) neurons in the RTN/pFRG serves as central chemoreceptor neurons and the CO2 sensitive Pre-I neurons express homeobox gene Phox2b. Phox2b encodes a transcription factor and is essential for the development of the sensory-motor visceral circuits. Mutations in human PHOX2B cause congenital hypoventilation syndrome, which is characterized by blunted ventilatory response to hypercapnia. Here we describe the generation of a novel transgenic (Tg) rat harboring fluorescently labeled Pre-I neurons in the RTN/pFRG. In addition, the Tg rat showed fluorescent signals in autonomic enteric neurons and carotid bodies. Because the Tg rat expresses inducible Cre recombinase in PHOX2B-positive cells during development, it is a potentially powerful tool for dissecting the entire picture of the respiratory neural network during development and for identifying the CO2/O2 sensor molecules in the adult central and peripheral nervous systems.

No MeSH data available.


Related in: MedlinePlus