Cytoarchitecture and CO2 Sensitivity of Phox2b-Positive Parafacial Neurons in the Newborn Rat Medulla

CHAPTER

Cytoarchitecture and CO2 Sensitivity of Phox2bPositive Parafacial Neurons in the Newborn Rat Medulla

4

Hiroshi Onimaru*,1, Keiko Ikeda{, Tani Mariho*, Kiyoshi Kawakami{ *

Department of Physiology, Showa University School of Medicine, Shinagawa-ku, Tokyo, Japan { Division of Biology, Hyogo College of Medicine, Nishinomiya, Hyogo, Japan { Division of Biology, Center for Molecular Medicine, Jichi Medical University, Shimotsuke, Tochigi, Japan 1 Corresponding author: Tel.: þ81-3-37848113; Fax: þ81-3-37840200, e-mail address: [email protected]

Abstract Preinspiratory (Pre-I) neurons in the parafacial respiratory group (pFRG) compose one of the respiratory rhythm generators in the medulla of the newborn rat. It has been shown that a subgroup of pFRG/Pre-I neurons could also work as central chemoreceptor neurons, because the CO2 sensitivity of these Pre-I neurons was preserved even after blockade of Naþ channels and Ca2þ channels, and the membrane depolarization induced by hypercapnic stimulation was mainly because of the closing of Kþ channels. These neurons, some of which were identified to be glutamatergic, express the transcription factor Phox2b. Phox2b expression was one of the most noticeable characteristics of pFRG/Pre-I neurons. We also found that Phox2b-expressing neurons in the parafacial region of the rostral ventral medulla tended to assemble around capillary blood vessels. In contrast, another subclass of the pFRG/Pre-I neurons was Phox2bnegative and CO2-insensitive. Some of these neurons were identified to be glycinergic or GABAergic. Thus, Phox2b expression is a key genetic marker that can be used to more clearly establish the cell architecture of the pFRG, which consists of heterogeneous neuronal subtypes. In this chapter, we elaborate on the CO2 sensitivity of Phox2b-positive/negative parafacial neurons and the cytoarchitecture in the newborn rat medulla, and discuss ionic mechanisms of CO2 responsiveness.

Keywords Phox2b, Glyt2, GAD, TASK, respiratory rhythm, central chemoreceptor, parafacial respiratory group, rostral ventrolateral medulla

Progress in Brain Research, Volume 209, ISSN 0079-6123, http://dx.doi.org/10.1016/B978-0-444-63274-6.00004-7 © 2014 Elsevier B.V. All rights reserved.

57

58

CHAPTER 4 Phox2b-Expressing Parafacial Neurons

1 INTRODUCTION The essential role of the respiratory center in homeostasis is to regulate ventilation so as to maintain optimal/healthy physiological arterial blood PCO2/pH and PO2. Such information is detected by peripheral and central chemoreceptors (Lahiri and Forster, 2003). The central chemoreceptors located in the lower brainstem are important in sensing mainly arterial blood CO2 concentration (or pH) (Guyenet et al., 2008; Nattie, 2001; Nattie and Li, 2009; Okada et al., 2001; Putnam, 2010; Richerson, 2004). Several hypotheses have been proposed with regard to the cellular mechanisms that could play the role of central chemoreceptors in respiratory control. They include the following: (1) The glutamatergic neuron theory involving the retrotrapezoid nucleus (RTN) posits that a group of glutamatergic neurons that express a transcription factor Phox2b are CO2/Hþ sensitive (Guyenet et al., 2010a,b); (2) the medullary serotonin (5-HT) neuron theory proposes that medullary (raphe) serotonin neurons are central chemoreceptors (Richerson, 2004); (3) the astrocyte–ATP theory proposes that ATP released from astrocytes in the ventral medulla acts as a mediator of central chemosensory transduction (Gourine et al., 2010); (4) the neuron/paraneuronal glia cell–acetylcholine theory posits that primary chemoreceptor cells (probably cholinergic) are located beneath large surface vessels within the marginal glial layer of the ventral medulla (Okada et al., 2002); and (5) multiple brainstem sites, including the previously mentioned chemoreceptors (i.e., distributed central chemoreceptor system), are synergistically involved in central chemoreception under various physiological conditions (Nattie and Li, 2009). In addition to the above mechanisms, it has been suggested that respiratory neurons in the medulla are directly sensitive to CO2/Hþ. For instance, 36% of preinspiratory (Pre-I) neurons in the rostral ventrolateral medulla corresponding to the caudal part of the parafacial respiratory group (pFRG) of the newborn rat are CO2 sensitive in the presence of tetrodotoxin (TTX) (Kawai et al., 2006). Pre-I neurons show pre-/postinspiratory bursting that is typically interrupted by inspiratoryrelated inhibition, and it has been suggested that they constitute one of the respiratory rhythm generators in the medulla (Ballanyi et al., 1999; Onimaru and Homma, 1992, 2003). We have shown more recently that Pre-I neurons of the pFRG were depolarized in response to high CO2 stimulation and that they expressed Phox2b, whereas Phox2b-negative Pre-I neurons were CO2-insensitive (Onimaru et al., 2008). Thus, it has been suggested that the pFRG includes a heterogeneous population of Pre-I neurons. The CO2 sensitivity of Phox2b-positive Pre-I neurons was preserved even after blockade of Naþ channels and Ca2þ channels, and the observed membrane depolarization was mainly caused by closure of Kþ channels (Onimaru et al., 2012a). These neurons are located in the vicinity of capillary blood vessels in the parafacial region of the ventral medulla (Onimaru and Dutschmann, 2012). In this chapter, we review the CO2 sensitivities of populations of Phox2b-positive and Phox2b-negative parafacial neurons and the cytoarchitecture within the newborn rat medulla, and discuss ionic mechanisms of CO2 responsiveness.

3 CO2 Sensitivity of pFRG/Pre-I Neurons

2 DISTRIBUTION OF pFRG/PRE-I NEURONS AND PHOX2B-EXPRESSING CELLS In the ventral medulla of the newborn rat, Phox2b-immunoreactive (-ir) cells are localized most densely in the ventrolateral medulla around the caudal end of the facial nucleus (caudal cluster of Phox2b-expressing cells) (Onimaru et al., 2008). In the more rostral medulla, Phox2b-expressing cells were found in the superficial area just ventral to the facial nucleus. At the level of the most rostral medulla, close to the rostral end of the facial nucleus, they formed one of the highest density clusters in the region ventrolateral to the facial nucleus (rostral cluster of Phox2b-expressing cells) (Fig. 1A). Such a distribution of Phox2b-expressing cells overlaps with the distribution of pFRG-Pre-I neurons, excluding the caudal area, where pFRG-Pre-I neurons are also found in the deeper ventral medulla (Fig. 1B). The distribution and characteristics of Phox2b-expressing cells in the parafacial region of the neonatal rat are basically consistent with those in the adult rat (Kang et al., 2007; Stornetta et al., 2006) and the neonatal mouse (Dubreuil et al., 2008). Thus, Phox2b-expressing pFRG neurons that are located close to the ventral surface overlap the RTN, at least partially, in adult rats (Stornetta et al., 2006). Moreover, the embryonic parafacial rhythm generator expressing Phox2b was suggested as a forerunner of neonatal pFRG (Thoby-Brisson et al., 2009).

3 CO2 SENSITIVITY OF pFRG/PRE-I NEURONS AND THEIR HISTOLOGICAL CHARACTERISTICS In newborn rats, the pFRG/Pre-I neurons located in the Phox2b-positive cell cluster are depolarized by high CO2 stimulation in the presence of TTX (Fig. 1). They are Phox2b-ir and strongly expressed neurokinin-1 receptor (NK1R) (Onimaru et al., 2008). Our studies have confirmed that some of the Phox2b-expressing/CO2-sensitive Pre-I neurons in the parafacial region are also VGlut2 mRNA positive, which indicated that they were glutamatergic (Table 1) (Onimaru et al., 2009). In addition, some are galanin-ir (T. Bautista and H. Onimaru, unpublished observation, see also Stronetta et al., 2009 for adult rats). These characteristics are similar to those of CO2sensitive Phox2b-positive neurons in the RTN of newborn mice (Dubreuil et al., 2008) and the adult rat (Guyenet et al., 2008; Stornetta et al., 2009; Takakura et al., 2008). Neurons with typical neonatal Pre-I firing patterns were not reported in the RTN of the adult rat, probably due to differences in experimental conditions and/or developmental stage (Guyenet et al., 2005; Stornetta et al., 2006). More recently, we confirmed that CO2 sensitivity of the pFRG/Pre-I neurons is preserved without any contribution of presynaptic mechanisms (Onimaru et al., 2012a). We found that ATP receptor and substance P receptor antagonists do not block membrane potential responses to hypercapnic stimulation (2% ! 8%) of pFRG/Pre-I neurons in the rostral parafacial region. Moreover, rostral pFRG/Pre-I

59

60

CHAPTER 4 Phox2b-Expressing Parafacial Neurons

A

B

Phox2b+ cells

Caudal

(a) –100 µm

pFRG-Pre-I neurons

(a) (-50 to -150 mm)

(–100 to –150 µm)

RFN

Rostral

RFN

CST

CST

(b) VIIc

(b) Vllc

(0 to –50 µm)

(-50 to +50 mm)

CST

CST

(c) +100 µm

(+50 to +100 µm)

(c) (+50 to +150 mm) FN

FN

CST

CST

(d) +400 µm

(d) (+150 to +450 mm)

(+350 to +400 µm)

FN

FN

CST

(e) +500 µm

(+450 to +500 µm)

CST

CST

(e) (+450 to +600 mm)

FN

0.5 mm

CST

FN

Phox2b +, CO2 + Phox2b -, CO2 Phox2b -, CO2 +

FIGURE 1 Phox2b-immunoreactive (-ir) cells and preinspiratory (Pre-I) neurons in the parafacial region of the neonatal rat medulla. (A) Distribution of Phox2b-ir cells. Each dot represents a single Phox2b-ir nucleus plotted on the 50-mm-thick coronal section. Phox2b-ir cells in neither the facial nor the retrofacial nuclei were plotted. Panels were arranged in a direction corresponding to the most caudal (upper) to most rostral (lower) sections. (B) Distribution of Pre-I neurons in the parafacial region (pFRG-Pre-I neurons). Location of neurons is plotted on 100–300 mm coronal sections. Values denote distance from the level of the caudal end of facial nucleus (VIIc) (Ruangkittisakul et al., 2006). Solid circles, Phox2b-immunoreactive and CO2-sensitive Pre-I neurons; open circles, Phox2b-negative and CO2-insensitive Pre-I neurons; open circles with center dot, Phox2b-negative and CO2-sensitive Pre-I neurons. FN, facial nucleus; RFN, retrofacial nucleus; CST, corticospinal tract. Modified from Onimaru et al. (2008).

neurons were depolarized by hypercapnia under conditions where the contribution of presynaptic components was inhibited (either in the presence of TTX and Cd2þ or in a low Ca2þ/high Mg2þ solution containing TTX and Cd2þ). A recent study by Gourine et al. (2010) showed strong evidence that ATP released from astrocytes in the chemoreceptor area of the ventral medulla acts as an essential factor for central chemosensory transduction. It was shown that the pH

3 CO2 Sensitivity of pFRG/Pre-I Neurons

Table 1 Characteristics of Pre-I neurons recorded in the caudal or rostral pFRG VGlut2 þ, Phox2b þ VGlut2 , Phox2b  Glyt2 þ, Phox2b  Glyt2 , Phox2b  Glyt2 , Phox2b þ GAD67 þ, Phox2b  GAD67 , Phox2b 

n

Location

7 6 3 4 1 3 3

Rostral (6), caudal (1) Caudal Caudal Caudal Caudal Caudal Caudal

GAD67, glutamate decarboxylase; Glyt2, glycine transporter 2; VGlut2, vesicular glutamate transporter 2. “þ” denotes that cells expressed each mRNA. “” denotes that cells did not express each mRNA. “Rostral” denotes that the Pre-I neurons were located in the rostral pFRG (400–500 mm rostral to the caudal end of the facial nucleus). “Caudal” denotes that the Pre-I neurons were located in the caudal pFRG (around the caudal end of the facial nucleus). Linearized plasmids were used for riboprobe templates. In situ hybridization was performed on 30-mm-thick 4% paraformaldehyde-fixed cryosections using single-stranded digoxigenin-UTP-labeled riboprobes (digoxigenin-UTP from Roche Diagnostics, Basel, Switzerland). Hybridization was performed at 50  C as described previously (Yokota et al., 2007). Signals were detected with an antidigoxigenin antibody-conjugated AP (Roche Diagnostics, Basel, Switzerland) and NBT/BCIP (Roche) for chromogen. The following probes were used: rat glycine transporter 2 (Glyt2), provided by Dr. Ikuko Tanaka; rat glutamate decarboxylase (GAD67), provided by Dr. Stanley Watson and Dr. Ilan Kerman. The plasmid for the VGlut2 riboprobe was provided by Drs. Ruth L. Stornetta and Shigefumi Yokota.

response of Phox2b-positive neurons in the RTN of organotypic slice cultures from rat pups (8–10 days old) was blocked by ATP receptor antagonists (MRS2179 and PPADS). Moreover, Wenker et al. (2010) suggested that astrocytes in the RTN sense CO2/Hþ in part through inhibition of a Kir4.1–Kir5.1-like current in slice preparations from 7- to 12-day-old rats. They also found that ATP receptor antagonists (PPADS and suramin) decreased CO2 sensitivity of RTN neurons and suggested that RTN astrocytes may provide an excitatory purinergic drive to pH-sensitive RTN neurons. In contrast, Mulkey et al. (2004) previously reported that the pH sensitivity of the RTN neurons in slices from 7- to 12-day-old rats was not blocked by PPADS. With regard to this apparent discrepancy, Wenker et al. (2010) suggested that the effects of ATP receptor antagonists in the previously mentioned studies depend on the buffers that are used (i.e., that the antagonists are less effective in HEPES buffer than in bicarbonate buffer). Although we used bicarbonate-buffered media and preparations from 0- to 4-day-old rats (Suzue, 1984), our results demonstrated that ATP receptor antagonists could not block membrane depolarization of pFRG/ RTN neurons induced by hypercapnia. The discrepancy may be due to differences among recording sites and experimental conditions as well as the different ages of the animals used in our study. In our experiments, with the exception of some cases in a low Ca2þ/high Mg2þ solution, all cases of membrane depolarization by hypercapnic stimulation were accompanied by an increase in input resistance. These pFRG/Pre-I neurons were predominantly Phox2b-ir. Our findings suggest that the response of these neurons to

61

62

CHAPTER 4 Phox2b-Expressing Parafacial Neurons

hypercapnia is induced by direct action on the postsynaptic membrane via the closing of Kþ channels (Onimaru et al., 2012a). We also showed that Phox2b-ir neurons in the parafacial region of the rostral ventral medulla tended to assemble around capillary blood vessels (Fig. 2; Onimaru et al., 2012b). In adult mice, ultrastructural studies have demonstrated that eGFP-labeled RTN neurons in Phox2b-eGFP transgenic mice were surrounded by capillaries and were often in direct contact with the basement membrane of capillaries (Lazarenko et al., 2009). Our findings are consistent with these observations in adult mice. In addition, we confirmed that pFRG/Pre-I neurons that were sensitive to hypercapnic stimulation in the presence of TTX were Phox2b-ir neurons and that they were tightly apposed to the blood vessels along the longitudinal axis (Onimaru et al., 2012b). Previous studies have reported a close relationship between chemosensitive neurons and blood vessels (Bradley et al., 2002; Okada et al., 2002). In particular, serotonergic neurons in the ventral medulla are tightly apposed to large midline penetrating arteries; patch clamp recordings from brain slices have confirmed that neurons with this anatomical specialization are chemosensitive (Bradley et al., 2002). Correspondingly, our findings showed that Phox2b-ir chemosensitive neurons in the parafacial regions were located close to capillary blood vessels (Onimaru et al., 2012b). In contrast, Phox2b-negative Pre-I neurons that were CO2-insensitive tended to be localized dorsally to the Phox2b-ir cell cluster at the level of the caudal end of the facial nucleus (Fig. 1; Onimaru et al., 2008). They did not tend to closely appose blood vessels in the manner observed in Phox2b-ir Pre-I neurons (i.e., along the longitudinal axis) (Onimaru et al., 2012b). The existence of such heterogeneous subpopulations with regard to CO2 responsiveness has been reported in a previous study (Kawai et al., 2006) in which Pre-I neurons were recorded in the caudal part of the pFRG in the newborn rat preparation. Previous electrophysiological studies suggested the presence of two types of Pre-I neurons; excitatory and inhibitory (Ballanyi et al., 1999; Onimaru and Homma, 1992). It remains to be studied whether the Phox2b-negative Pre-I neurons in the neonatal rat preparation are glycinergic, GABAergic, adrenergic, or glutamatergic. We have thus far confirmed that Pre-I neurons recorded in the pFRG are either tyrosine hydroxylase-negative (Onimaru et al., 2008) or phenylethanolamine N-methyltransferase-negative (H. Onimaru, K. Ikeda and K. Kawakami, unpublished observation), indicating that they were not adrenergic, regardless of their Phox2b status. Moreover, some of the Phox2bnegative Pre-I neurons were identified as GABAergic or glycinergic because of their expression of glutamic acid decarboxylase 67 (GAD67) or glycine transporter 2 (GLYT2)-mRNA (Fig. 3 and Table 1). Although we have found no VGlut2-positive Pre-I neurons that were Phox2b-negative to date, some of Phox2b-negative Pre-I neurons may be excitatory. Moreover, we have found that inspiratory and expiratory neurons in the caudal part of the pFRG were Phox2b-negative (Onimaru et al., 2009). Fortuna et al. (2008) reported that a population of rostral ventral respiratory neurons (i.e., Bo¨tzinger glycinergic expiratory augmenting neurons) develops a preinspiratory discharge during hypercapnic hypoxia in in vivo preparations of adult rats. A subgroup of Pre-I neurons in the newborn preparation may correspond to such

3 CO2 Sensitivity of pFRG/Pre-I Neurons

FIGURE 2 Distribution of blood vessels and Phox2b cells in the rostral ventral medulla of a 1-day-old rat. The blood vessels stained by neurobiotin (Onimaru et al., 2012b) are shown in green and Phox2b-positive nuclei are indicated in white. Bottom: higher magnification of the highlighted square region in the top image. Note that Phox2b-positive cells are assembled around capillary blood vessels. FN, facial nucleus; D, dorsal; M, medial.

63

64

CHAPTER 4 Phox2b-Expressing Parafacial Neurons

A

C

mV -40 -50 0.1 mV

Vm C4 5s

B Vm

D

8% CO2

1 min 10 mV

D¢

FIGURE 3 Example of Glyt2-mRNA positive Pre-I neurons in the caudal parafacial region. The Pre-I neuron was recorded at the level of 50 mm caudal to the caudal end of the facial nucleus. (A) Membrane potential trajectory and C4 inspiratory activity. (B) Change in membrane potential in response to hypercapnia in the presence of 0.5 mM TTX. CO2 concentration was increased from 2% to 8%. Square current pulse (0.1 Hz, 20 pA) was applied to monitor the change of input resistance. The negative deflections of the baseline membrane potential are proportional to the input resistance. Note that application of 8% CO2 did not induce changes in membrane potential and input resistance. (C) Glyt2-mRNA expression. The neuron labeled with Lucifer Yellow in the electrode solution, visualized with Alexa Fluor 488 (green). (D, D0 ) Higher magnification of the cell. Note that this cell (green in D) expressed Glyt2 mRNA (an arrow, D0 ). This cell did not show Phox2b immunoreactivity.

inhibitory neurons in the adult rat (see also Ezure et al., 2003). Although there were differences in experimental conditions between these studies, the results also support the existence of heterogeneous subpopulations of Pre-I neurons in the rostral medulla around the caudal end of the facial nucleus. In conclusion, Phox2b expression more clearly establishes the cell architecture of the pFRG. Phox2b expression is the most noticeable characteristic of subtypes of CO2-sensitive pFRG/Pre-I neurons, whereas the genetic markers of CO2-insensitive pFRG/Pre-I neurons are unknown.

4 Ionic Mechanisms of CO2 Sensitivity

4 IONIC MECHANISMS OF CO2 SENSITIVITY It has been suggested that membrane responsiveness to CO2/Hþ stimulation is generally due to the closing of Kþ channels (Jiang et al., 2005; Putnam et al., 2004). Indeed, membrane depolarization in response to hypercapnia accompanies an increase in input resistance in parafacial neurons, consistent with the involvement of potassium channels, as suggested in previous studies (Guyenet et al., 2008; Kawai et al., 2006). Although the types of potassium channels that are involved in this response remain unknown, acid-sensitive TASK channels might be involved in the CO2/Hþ responses as the Kþ channels. On the basis of analyses of TASK1/3 knockout mice, Mulkey et al. (2007) suggested that these potassium channels are not involved in chemoreception of RTN neurons, but that they are necessary for chemoreception of raphe neurons. Gestreau et al. (2010) reported the presence of beta-galactosidase expressed from the knock-in LacZ allele targeted the TASK2 gene locus in the pFRG/RTN neurons, implying that pFRG/RTN neurons are derived from cells that had once expressed the LacZ gene. Interestingly, all TASK2-positive RTN neurons were lost in mice bearing a Phox2b mutation that causes human congenital central hypoventilation syndrome (Gestreau et al., 2010). Thus, TASK2 channels could be the potassium channel responsible for CO2/Hþ responses. However, since Task2 / mice showed hypersensitivity to low CO2 concentrations (Gestreau et al., 2010), the results seem to exclude a primary role of TASK2 channels as CO2 sensors. Moreover, the relatively weak expression of mRNA for TASK channels in pFRGPhox2b neurons in our study (Figs. 4 and 5) is consistent with previous results indicating a minor contribution to CO2/Hþ responses in the pFRG/RTN neurons. In addition to TASK channels, conductance of many potassium channels changes in response to changing pH (Jiang et al., 2005). In many cases, these potassium channels close in response to reduced pH (Putnam et al., 2004), but responsible potassium channels seem to differ among different central chemoreceptors (Putnam, 2010). Involvement of Ca2þ channels or nonselective cation channels (CAN) in CO2/pH responses has also been suggested (Putnam, 2010). In our recent studies, many pFRG-Pre-I neurons produced Ca2þ action potentials in the presence of TTX (e.g., Fig. 6C), whereas depolarizing responses to high CO2 stimulation are known to depend on a decrease in potassium conductance and not on an increase in Ca2þ conductance (Onimaru et al., 2012a). Our data suggest that the activation of Ca2þ channels may facilitate membrane depolarization in response to high CO2/Hþ stimulation. Kawai et al. (2006) showed that approximately 20% of CO2/Hþ-sensitive neurons depolarized without a change in input resistance and that the response was not affected by potassium channel blockers (Kawai et al., 2006). Our recent study also demonstrated the existence of similar types of pFRG-Pre-I neurons (Onimaru et al., 2012a). Unknown mechanisms in addition to ionic channels could be involved in these depolarizing responses. Transporter or electrogenic pump mechanisms are possible candidates and a topic for future study (Putnam, 2010). In terms of CO2/Hþ chemoreception, Hþ is the most important molecule to affect Kþ channels, whereas the existence of any system that directly senses the CO2 molecule still remains to be found.

65

66

CHAPTER 4 Phox2b-Expressing Parafacial Neurons

FIGURE 4 Expression of TASK1-, 2-, and 3-mRNA in the facial nucleus by in situ hybridization. (A) TASK1-mRNA expressions are detected in the facial nucleus. (B) TASK2-mRNA expressions are detected in the facial motor neurons. Some expression is also detected in the parafacial region ventral to the facial motor neurons. (C) TASK3-mRNA expression is clearly detected in the facial motor neurons. FN, facial nucleus; D, dorsal; M, medial. Partial cDNAs of mouse potassium channel, subfamily K, member 5, Kcnk5 (TASK2; nucleotide number 1358–2219, NM_021542), rat Kcnk3 (TASK1; nucleotide number 814–1339, NM_033376), and rat Kcnk9 (TASK3; nucleotide number 2582–3186, DQ897665) were obtained by RT-PCR with total RNA of mouse or rat adult brains (Clontech Lab. Inc.). They were subcloned into pGEM-T Easy Vector (Promega, Madison, WI) and confirmed by sequencing. The region of mouse Kcnk5 used for the riboprobe has 91% homology with rat Kcnk5 (NM_001039516).

5 Conclusion

FIGURE 5 TASK3-mRNA and Phox2b expression in the parafacial region. TASK3-mRNAs are strongly expressed in the facial motor neurons, but the expression in Phox2b-ir cells in the parafacial region (pink; surrounded by a dotted line) is weak. FN, facial nucleus.

When the CO2 molecule is diffused close to or within chemoreceptor cells, Hþ is produced by carbonic anhydrase and this change may be detected by acid-sensitive Kþ channels (Necakov et al., 2002; Torrance, 1993). Possible sites for Hþ production may be intra- or extracellularly located, because carbonic anhydrase can function within the intracellular environment and extracellularly in the membrane-bound form (Parkkila et al., 2000; Putnam, 2010). In this context, it would be interesting to investigate how facial motor neurons respond to high CO2 or low pH environments. The facial motor neurons express TASK1, TASK2, and TASK3 channels (Figs. 4 and 5), whereas Gestreau et al. (2010) showed no expression of the TASK2 gene replaced by LacZ (knock-in) in the facial nucleus. The facial motor neurons responded with membrane depolarization to low pH stimulation, but with high CO2 stimulation they responded with membrane hyperpolarization to high (Fig. 6A and B). In contrast, pFRG-Pre-I neurons responded with membrane depolarization to both stimuli (Fig. 6C). Although membrane-bound carbonic anhydrase might be involved in these responses of pFRG-Pre-I neurons, this has yet to be confirmed.

5 CONCLUSION The pFRG/Pre-I neurons consist of heterogeneous subpopulations with regard to CO2 responsiveness and Phox2b-immunoreativity. CO2-sensitive Pre-I neurons are Phox2b-ir and include glutamatergic, NK1R-expressing, and galanin-ir cells. CO2-insensitive Pre-I neurons are Phox2b-negative and include GABAergic and glycinergic cells. Whether CO2-insensitive Pre-I neurons include glutamatergic cells is

67

68

CHAPTER 4 Phox2b-Expressing Parafacial Neurons

FIGURE 6 CO2/Hþ responses of the facial motor neuron compared to those of the pFRG neuron. (A) Membrane potential response of a facial motor neuron to lowering pH at constant CO2 (5%) concentration in the presence of 0.5 mM TTX. Square current pulse (0.1 Hz, 60 pA in A and B) was applied to monitor the change of input resistance. The negative deflections of the baseline membrane potential are proportional to the input resistance. Note that low pH solution induced membrane depolarization (þ1 to þ3 mV, n ¼ 3/4). (B) Membrane potential response of a facial motor neuron to increasing CO2 concentration (2% ! 8%) at 26 mM NaHCO3 in the presence of 0.5 mM TTX. Note that high CO2 solution induced membrane hyperpolarization (1 to 5 mV, n ¼ 6/8). (C) An example of membrane potential response of pFRG-Pre-I neuron to an increase in CO2 concentration (2% ! 8%) at 26 mM NaHCO3 in the presence of 0.5 mM TTX. Note strong depolarization with increase in the input resistance (Onimaru et al., 2008). Ca2þ action potentials are induced during the membrane depolarization by hypercapnia.

not clear. CO2 sensitivity is primarily linked to closure of the potassium channels of the postsynaptic membrane and future studies could provide important clarification on the types of potassium channels involved in this response. Phox2b-ir neurons in the parafacial region of the rostral ventral medulla tended to be located around capillary blood vessels. The location of Phox2b-ir neurons, including pFRG/Pre-I neurons, is appropriate to their physiological role of sensing blood CO2 concentration.

Acknowledgments We thank Drs. Ruth L. Stornetta and Shigefumi Yokota for providing the VGlut2 probe plasmid, Dr. Ikuko Tanaka for the Glyt2 probe plasmid, and Drs. Stanley Watson and Ilan Kerman for the GAD probe plasmid. This work was supported by the Grants-in Aid for Scientific Research (KAKENHI: 19500277, 22500296).

References

References Ballanyi, K., Onimaru, H., Homma, I., 1999. Respiratory network function in the isolated brainstem-spinal cord of newborn rats. Prog. Neurobiol. 59, 583–634. Bradley, S.R., Pieribone, V.A., Wang, W., Severson, C.A., Jacobs, R.A., Richerson, G.B., 2002. Chemosensitive serotonergic neurons are closely associated with large medullary arteries. Nat. Neurosci. 5, 401–402. Dubreuil, V., Ramanantsoa, N., Trochet, D., Vaubourg, V., Amiel, J., Gallego, J., Brunet, J.F., Goridis, C., 2008. A human mutation in Phox2b causes lack of CO2 chemosensitivity, fatal central apnea, and specific loss of parafacial neurons. Proc. Natl. Acad. Sci. U.S.A. A105, 1067–1072. Ezure, K., Tanaka, I., Kondo, M., 2003. Glycine is used as a transmitter by decrementing expiratory neurons of the ventrolateral medulla in the rat. J. Neurosci. 23, 8941–8948. Fortuna, M.G., West, G.H., Stornetta, R.L., Guyenet, P.G., 2008. Botzinger expiratoryaugmenting neurons and the parafacial respiratory group. J. Neurosci. 28, 2506–2515. Gestreau, C., Heitzmann, D., Thomas, J., Dubreuil, V., Bandulik, S., Reichold, M., Bendahhou, S., Pierson, P., Sterner, C., Peyronnet-Roux, J., Benfriha, C., Tegtmeier, I., Ehnes, H., Georgieff, M., Lesage, F., Brunet, J.F., Goridis, C., Warth, R., Barhanin, J., 2010. Task2 potassium channels set central respiratory CO2 and O2 sensitivity. Proc. Natl. Acad. Sci. U.S.A. A107, 2325–2330. Gourine, A.V., Kasymov, V., Marina, N., Tang, F., Figueiredo, M.F., Lane, S., Teschemacher, A.G., Spyer, K.M., Deisseroth, K., Kasparov, S., 2010. Astrocytes control breathing through pH-dependent release of ATP. Science 329, 571–575. Guyenet, P.G., Mulkey, D.K., Stornetta, R.L., Bayliss, D.A., 2005. Regulation of ventral surface chemoreceptors by the central respiratory pattern generator. J. Neurosci. 25, 8938–8947. Guyenet, P.G., Stornetta, R.L., Bayliss, D.A., 2008. Retrotrapezoid nucleus and central chemoreception. J. Physiol. 586, 2043–2048. Guyenet, P.G., Stornetta, R.L., Abbott, S.B., Depuy, S.D., Fortuna, M.G., Kanbar, R., 2010a. Central CO2 chemoreception and integrated neural mechanisms of cardiovascular and respiratory control. J. Appl. Physiol. 108, 995–1002. Guyenet, P.G., Stornetta, R.L., Bayliss, D.A., 2010b. Central respiratory chemoreception. J. Comp. Neurol. 518, 3883–3906. Jiang, C., Rojas, A., Wang, R., Wang, X., 2005. CO2 central chemosensitivity: why are there so many sensing molecules? Respir. Physiol. Neurobiol. 145, 115–126. Kang, B.J., Chang, D.A., Mackay, D.D., West, G.H., Moreira, T.S., Takakura, A.C., Gwilt, J.M., Guyenet, P.G., Stornetta, R.L., 2007. Central nervous system distribution of the transcription factor Phox2b in the adult rat. J. Comp. Neurol. 503, 627–641. Kawai, A., Onimaru, H., Homma, I., 2006. Mechanisms of CO2/H þ chemoreception by respiratory rhythm generator neurons in the medulla from newborn rats in vitro. J. Physiol. 572, 525–537. Lahiri, S., Forster 2nd., R.E., 2003. CO2/H(þ) sensing: peripheral and central chemoreception. Int. J. Biochem. Cell Biol. 35, 1413–1435. Lazarenko, R.M., Milner, T.A., Depuy, S.D., Stornetta, R.L., West, G.H., Kievits, J.A., Bayliss, D.A., Guyenet, P.G., 2009. Acid sensitivity and ultrastructure of the retrotrapezoid nucleus in Phox2b-EGFP transgenic mice. J. Comp. Neurol. 517, 69–86. Mulkey, D.K., Stornetta, R.L., Weston, M.C., Simmons, J.R., Parker, A., Bayliss, D.A., Guyenet, P.G., 2004. Respiratory control by ventral surface chemoreceptor neurons in rats. Nat. Neurosci. 7, 1360–1369.

69

70

CHAPTER 4 Phox2b-Expressing Parafacial Neurons

Mulkey, D.K., Talley, E.M., Stornetta, R.L., Siegel, A.R., West, G.H., Chen, X., Sen, N., Mistry, A.M., Guyenet, P.G., Bayliss, D.A., 2007. TASK channels determine pH sensitivity in select respiratory neurons but do not contribute to central respiratory chemosensitivity. J. Neurosci. 27, 14049–14058. Nattie, E.E., 2001. Central chemosensitivity, sleep, and wakefulness. Respir. Physiol. 129, 257–268. Nattie, E., Li, A., 2009. Central chemoreception is a complex system function that involves multiple brain stem sites. J. Appl. Physiol. 106, 1464–1466. Necakov, A., Peever, J.H., Shen, L., Duffin, J., 2002. Acetazolamide and respiratory chemosensitivity to CO(2) in the neonatal rat transverse medullary slice. Respir. Physiol. Neurobiol. 132, 279–287. Okada, Y., Chen, Z., Kuwana, S., 2001. Cytoarchitecture of central chemoreceptors in the mammalian ventral medulla. Respir. Physiol. 129, 13–23. Okada, Y., Chen, Z., Jiang, W., Kuwana, S., Eldridge, F.L., 2002. Anatomical arrangement of hypercapnia-activated cells in the superficial ventral medulla of rats. J. Appl. Physiol. 93, 427–439. Onimaru, H., Dutschmann, M., 2012. Calcium imaging of neuronal activity in the most rostral parafacial respiratory group of the newborn rat. J. Physiol. Sci. 62, 71–77. Onimaru, H., Homma, I., 1992. Whole cell recordings from respiratory neurons in the medulla of brainstem-spinal cord preparations isolated from newborn rats. Pflugers Arch. 420, 399–406. Onimaru, H., Homma, I., 2003. A novel functional neuron group for respiratory rhythm generation in the ventral medulla. J. Neurosci. 23, 1478–1486. Onimaru, H., Ikeda, K., Kawakami, K., 2008. CO2-sensitive preinspiratory neurons of the parafacial respiratory group express Phox2b in the neonatal rat. J. Neurosci. 28, 12845–12850. Onimaru, H., Ikeda, K., Kawakami, K., 2009. Phox2b, RTN/pFRG neurons and respiratory rhythmogenesis. Respir. Physiol. Neurobiol. 168, 13–18. Onimaru, H., Ikeda, K., Kawakami, K., 2012a. Postsynaptic mechanisms of CO2 responses in parafacial respiratory neurons of newborn rats. J. Physiol. 590 (7), 1615–1624. Onimaru, H., Ikeda, K., Kawakami, K., 2012b. Relationship between the distribution of the paired-like homeobox gene (Phox2b) expressing cells and blood vessels in the parafacial region of the ventral medulla of neonatal rats. Neuroscience 212, 131–139. Parkkila, S., Parkkila, A.K., Saarnio, J., Kivela, J., Karttunen, T.J., Kaunisto, K., Waheed, A., Sly, W.S., Tureci, O., Virtanen, I., Rajaniemi, H., 2000. Expression of the membraneassociated carbonic anhydrase isozyme XII in the human kidney and renal tumors. J. Histochem. Cytochem. 48, 1601–1608. Putnam, R.W., 2010. CO2 chemoreception in cardiorespiratory control. J. Appl. Physiol. 108, 1796–1802. Putnam, R.W., Filosa, J.A., Ritucci, N.A., 2004. Cellular mechanisms involved in CO(2) and acid signaling in chemosensitive neurons. Am. J. Physiol. Cell Physiol. 287, C1493–C1526. Richerson, G.B., 2004. Serotonergic neurons as carbon dioxide sensors that maintain pH homeostasis. Nat. Rev. Neurosci. 5, 449–461. Ruangkittisakul, A., Schwarzacher, S.W., Secchia, L., Poon, B.Y., Ma, Y., Funk, G.D., Ballanyi, K., 2006. High sensitivity to neuromodulator-activated signaling pathways at physiological [K þ] of confocally imaged respiratory center neurons in on-line-calibrated newborn rat brainstem slices. J. Neurosci. 26, 11870–11880.

References

Stornetta, R.L., Moreira, T.S., Takakura, A.C., Kang, B.J., Chang, D.A., West, G.H., Brunet, J.F., Mulkey, D.K., Bayliss, D.A., Guyenet, P.G., 2006. Expression of Phox2b by brainstem neurons involved in chemosensory integration in the adult rat. J. Neurosci. 26, 10305–10314. Stornetta, R.L., Spirovski, D., Moreira, T.S., Takakura, A.C., West, G.H., Gwilt, J.M., Pilowsky, P.M., Guyenet, P.G., 2009. Galanin is a selective marker of the retrotrapezoid nucleus in rats. J. Comp. Neurol. 512, 373–383. Suzue, T., 1984. Respiratory rhythm generation in the in vitro brain stem-spinal cord preparation of the neonatal rat. J. Physiol. 354, 173–183. Takakura, A.C., Moreira, T.S., Stornetta, R.L., West, G.H., Gwilt, J.M., Guyenet, P.G., 2008. Selective lesion of retrotrapezoid Phox2b-expressing neurons raises the apnoeic threshold in rats. J. Physiol. 586, 2975–2991. Thoby-Brisson, M., Karlen, M., Wu, N., Charnay, P., Champagnat, J., Fortin, G., 2009. Genetic identification of an embryonic parafacial oscillator coupling to the preBo¨tzinger complex. Nat. Neurosci. 12, 1028–1035. Torrance, R.W., 1993. Carbonic anhydrase near central chemoreceptors. Adv. Exp. Med. Biol. 337, 235–239. Wenker, I.C., Kreneisz, O., Nishiyama, A., Mulkey, D.K., 2010. Astrocytes in the retrotrapezoid nucleus sense Hþ by inhibition of a Kir4.1-Kir5.1-like current and may contribute to chemoreception by a purinergic mechanism. J. Neurophysiol. 104, 3042–3052. Yokota, S., Oka, T., Tsumori, T., Nakamura, S., Yasui, Y., 2007. Glutamatergic neurons in the Kolliker-Fuse nucleus project to the rostral ventral respiratory group and phrenic nucleus: a combined retrograde tracing and in situ hybridization study in the rat. Neurosci. Res. 59, 341–346.

71