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Symposium Reports |
1 Division of Molecular Neurobiology, National Institute for Basic Biology and School of Life Science, Graduate University for Advanced Studies, Okazaki 444-8787, Japan
Abstract
Dehydration causes an increase in the sodium (Na) concentration and osmolarity of body fluids. For Na homeostasis of the body, control of Na and water intake and excretion are of prime importance. Although the circumventricular organs (CVOs) were suggested to be involved in body-fluid homeostasis, the system for sensing Na levels within the brain, which is responsible for the control of Na- and water-intake behaviour, has long been an enigma. Nax is an atypical sodium channel that is assumed to be a descendant of the voltage-gated sodium channel family. Our studies on the Nax-gene-targeting (Nax–/–) mouse revealed that Nax channels are localized to the CVOs and serve as a sodium-level sensor of body fluids. As the first step to understand the cellular mechanism by which the information sensed by Nax channels is reflected in the activity of the organs, we dissected the subcellular distribution of Nax. Double-immunostaining and immuno-electron microscopic analyses revealed that Nax is exclusively localized to perineuronal lamellate processes extending from ependymal cells and astrocytes in the organs. In addition, glial cells isolated from the subfornical organ were sensitive to an increase in the extracellular sodium level, as analysed by an ion-imaging method. These results suggest that glial cells bearing Nax channels are the first to sense a physiological increase in the level of sodium in body fluids, and regulate the neural activity of the CVOs by enveloping neurons. Close communication between inexcitable glial cells and excitable neural cells thus appears to be the basis of the central control of salt homeostasis.
(Received 11 January 2007;
accepted after revision 28 February 2007; first published online 9 March 2007)
Corresponding author M. Noda: Division of Molecular Neurobiology, National Institute for Basic Biology, 5-1 Higashiyama, Myodaiji-cho, Okazaki 444-8787, Japan. Email: madon{at}nibb.ac.jp
Sodium is a major electrolyte of extracellular fluids and the main determinant of osmolarity. Since sodium homeostasis is essential to life, sodium-ion (Na+) concentrations in plasma and cerebrospinal fluid (CSF) are continuously monitored to maintain a physiological level of sodium in body fluids. A specific sodium sensor has been long hypothesized to exist in the brain for the control of sodium intake (Weisinger et al. 1979; Denton et al. 1996) as well as natriuresis (Cox et al. 1987; Denton et al. 1996). The site for the sensing was postulated to be in the circumventricular organs (CVOs) in the periventricular region of the brain (Cox et al. 1987; Park et al. 1989; Denton et al. 1996). The CVOs, mid-line structures found in the brain of all vertebrates (McKinley et al. 2003), are so named because of their proximity to the ventricles of the brain. Their specialized common features are extensive vascularization, no blood–brain barrier and atypical ependymal cells. Among the CVOs, only three loci, the subfornical organ (SFO), organum vasculosum of the lamina terminalis (OVLT) and area postrema (AP), harbour neuronal cell bodies that have efferent neural connections to many other areas of the brain. Their neurons are supposedly exposed to the chemical environment of the general circulation, unlike other neuronal perikarya in the CNS, because of the lack of a normal blood–brain barrier. Therefore, these three CVOs are termed sensory circumventricular organs (Johnson & Gross, 1993).
We previously found that Nax, an atypical sodium channel whose structure is poorly homologous (
50% identical) to the voltage-gated sodium channels (more than 80% identical to each other; Fig. 1A; Goldin et al. 2000), is expressed in four CVOs: the SFO, OVLT, median eminence (ME) and posterior pituitary (Watanabe et al. 2000). Nax–/– mice showed increased neural activity in the SFO and OVLT after water deprivation compared with wild-type (Nax+/+) mice as estimated from Fos immunoreactivity (Watanabe et al. 2000). Moreover, Nax–/– mice did not stop ingesting salt when dehydrated, while Nax+/+ mice avoided salt (Watanabe et al. 2000; Hiyama et al. 2004). Subsequently, we demonstrated that the Nax channel is a concentration-sensitive sodium channel with a threshold value of approximately 150 mM for the extracellular sodium ion (Hiyama et al. 2002). We further showed that salt-aversive behaviour does not occur on direct infusion of a hypertonic sodium solution into the cerebral ventricle in Nax–/– mice, in contrast to Nax+/+ mice (Hiyama et al. 2004). The behavioural phenotype of Nax–/– mice was completely recovered by a site-directed transfer of the Nax gene with an adenoviral vector into the SFO (Hiyama et al. 2004). All these findings indicate that the Nax channel is the brain sodium-level sensor that was postulated to be present in the CVOs involved in the regulation of salt intake (Andersson, 1978).
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Targeted disruption of the Nax gene
The lacZ gene was designed to be inserted in-frame into the protein-coding exon 1 of the Nax gene. The N-terminal 20-amino-acid sequence of Nax was fused with ß-galactosidase. The original genomic structure of the Nax gene was not modified in the targeting vector except for the insertion of the lacZ-neo cassette (Fig. 1B) to make sure that the lacZ gene is expressed in place of the Nax gene in the targeted mice. Analysis of the lacZ expression clearly demonstrated that Nax is expressed in the four CVOs and several minor nuclei in the CNS (Fig. 1C; see Watanabe et al. 2000).
Nax-knockout mice do not stop ingesting salt when dehydrated
To analyse the daily pattern of water and salt intake in a free-moving state, we developed a drinking-behaviour-monitoring system for mice (Fig. 2A). Using this system, we measured the amount drunk by individual mice (Hiyama et al. 2004). Then the preference ratios, defined as the volume ratio of saline intake divided by saline plus pure water intake in 12 h, were calculated (Fig. 2B). When fully satiated with water, both the Nax–/– and Nax+/+ mice showed a progressive aversion at concentrations above 0.3 M. The preference–aversion curves for a series of NaCl solutions was nearly identical between the two genotypes, indicating that Nax–/– mice have a normal tasting ability for salt (see also Watanabe et al. 2000).
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Sodium concentrations in plasma and the CSF increase by 5–10% during thirsty conditions (Wakerley et al. 1978; Nose et al. 1992). In our dehydration experiments for 2 days, the sodium concentration in the blood increased from 148.2 ± 3.3 (n = 8) to 176.3 ± 12.2 mM in Nax+/+ mice, and from 146.1 ± 2.7 to 172.3 ± 9.0 mM in Nax–/– mice. Thus, Nax–/– mice appear to be deficient in the ability to reset the threshold in the brain that is required when dehydrated. The difference between the two genotypes was greatest at 0.3 M NaCl in the dehydrated condition.
Using the same system, we monitored the time course of intake of pure water and 0.3 M NaCl independently. After dehydration, animals of both genotypes rushed to drink fluids in large amounts, and subsequently the drinking rate decreased gradually (Fig. 2C). When dehydrated, Nax+/+ mice preferred pure water and avoided the hypertonic saline and, as a result, the preference ratio for 0.3 M NaCl was markedly reduced. In contrast, Nax–/– mice took both pure water and 0.3 M NaCl equally, and the preference ratio for 0.3 M NaCl was not changed by dehydration (Fig. 2D). In spite of this difference, the time course profiles of the total volume drunk (the total amount of pure water plus 0.3 M NaCl) were not significantly different between the two genotypes, not only before but also after the water deprivation (Fig. 2E).
Furthermore, under the acute salt appetite condition induced by intraperitoneal injection of a diuretic drug (0.6 mg furosemide in 0.12 ml normal saline), with a sodium-depleted diet, the Nax–/– mice showed an approximately twofold increase in the ingestion of 0.3 M NaCl (1.2 ml per 2 h) compared with the wild-type (Nax+/+; 0.5 ml per 2 h) and heterozygous mutant mice (Nax+/–; 0.6 ml per 2 h). This abnormal ingestion of hypertonic saline stopped when a sodium-containing conventional food was provided (0.2–0.3 ml per 2 h; Watanabe et al. 2000).
Nax is a sodium concentration-sensitive sodium channel
These findings led us to speculate that Nax is directly involved in the sodium-level sensing mechanism in the brain. We verified this possibility by imaging analysis using a sodium-sensitive dye (Hiyama et al. 2002). When the extracellular sodium-ion concentration ([Na+]o) was increased from the control value of 145 mM (physiological level) to 170 mM by bath application, the intracellular sodium-ion concentration ([Na+]i) of some cells dissociated from the SFO of Nax+/+ mice showed a pronounced increase (Fig. 3A and B). Importantly, all the [Na+]i-responsive cells were Nax-immunoreactive. These cells responded to the rise in [Na+]o, but not to the rise in osmolarity or extracellular chloride-ion concentration ([Cl–]o; Fig. 3C). Tetrodotoxin (TTX) at 1 mM did not antagonize the response (Fig. 3C). Extracellular [Na+] at half-maximal (C1/2) was 157 mM (Fig. 3D).
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Nax-knockout mice show abnormal salt-intake behaviour during the microinjection of hypertonic NaCl into the cerebral ventricle
In dehydrated animals, the Na+ concentration of body fluids is obviously inclined to rise all over the body. In this context, it is important to examine the effects of the direct stimulation of CVOs with hypertonic Na+ solutions from the cerebral ventricle on drinking behaviour in the two-bottle test (Fig. 4A). When isotonic saline solution (0.145 M NaCl) was continuously infused into the ventricle (0.013 µl min–1), both genotypes took 0.3 M NaCl and water equivalently (Fig. 4C), indicating that neither the operation nor the injection itself affected drinking behaviour.
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In contrast, when a hypertonic mannitol solution in isotonic saline (0.145 M NaCl + 0.71 M mannitol; the osmotic pressure being approximately equivalent to 0.5 M NaCl) was continuously infused into the ventricle, both genotypes took 0.3 M NaCl and water equivalently (Fig. 4B and C). Importantly, the total intake volume was increased equally in both genotypes when mice were infused with the hypertonic saline solution and hypertonic mannitol solution (Fig. 4D), suggesting that the increase in total intake is regulated by the osmolarity of the CSF. These results indicate that Nax is involved in sensing an increase in the level of Na+ in the CSF, specifically relevant to the control of NaCl intake. In contrast, the control of the total intake is dependent on the osmolarity, and independent of Nax. Here, it should be noted that Liedtke & Friedman (2003) and Ciura & Bourque (2006) recently suggested that the transient receptor potential vanilloid type 4 (Trpv4) and a variant of Trpv1, respectively, expressed in the OVLT, are involved in systemic osmosensing.
Abnormal salt-intake behaviour of Nax-knockout mice is rescued by transduction of the Nax gene into the SFO
To specify the brain locus from which the difference in Na+- and water-intake behaviour in the two genotypes originates, the Nax gene was locally introduced into the brain of Nax–/– mice with an adenoviral expression vector carrying Nax under the control of the cytomegalovirus promoter. An adenoviral expression vector encoding the enhanced green fluorescent protein (EGFP) gene (egfp) was co-injected to identify the infected site after the experiments. One week after the injection, the mice were subjected to behavioural tests to examine whether the salt-aversion behaviour was restored. Mice that had received an injection of the Nax-adenoviral vector into the SFO showed an aversion to salt similar to Nax+/+ mice when dehydrated (Fig. 5). Here, the intake of 0.3 M NaCl was decreased and that of water was increased by the Nax gene transduction, the total intake again being constant.
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Nax channels are expressed in glial lamellate processes
To identify the cellular population expressing Nax channels in the SFO and OVLT, together with the subcellular distribution of the channels, we performed immuno-electron microscopic experiments using Nax-antibody. Optional microscopic immunostaining of the SFO is shown in Fig. 6A and B. The most intensive signals appeared to be associated with the periphery of a subset of neural cell bodies and their principal processes. Figure 6C–F shows immuno-electron micrographs of the localization of Nax in the SFO. Here, immunopositive signals turned out to be localized mainly to thin lamellar processes (arrows) surrounding neuronal cell bodies and processes and, in some cases, synapses. These immunopositive thin lamellate processes were apparently extended from cell bodies of ependymal cells (Fig. 6C and D) or astrocytes (Fig. 6E and F). Nax channels thus appeared to be transported and localized to the lamellar processes and endfeet of ependymal cells and astrocytes surrounding neurons (Fig. 6G). In the OVLT, similar features were observed, in that endfeet of ependymal cells and astrocytes surrounding neural cell bodies and neurites were positive for Nax (Watanabe et al. 2006). We could not detect significant immunopositive signals in neuronal cell bodies and their processes, including synapses, in either the SFO or the OVLT.
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We next examined what types of neurons are surrounded by Nax-positive lamellate processes of glia. Numerous glutamatergic, serotonergic, GABAergic and glycinergic fibres and their terminals have been identified by immunohistochemical techniques in the sensory CVOs (McKinley et al. 2003). However, identified neuronal cell bodies containing neurotransmitters in the SFO are only GABAergic interneurons. Therefore, we examined the relationship between GABAergic interneurons and Nax-positive glial cells using GAD-GFP knock-in mice (Tamamaki et al. 2003).
As shown in Fig. 8A-C, some GAD-positive neurons appeared to be surrounded by Nax-positive glial processes in the SFO. In contrast, Nax-positive glial processes extended also to areas where GAD-positive neurons were not observed. This observation was confirmed by electron microscopic analysis (Watanabe et al. 2006). In the OVLT region, in contrast, GAD-positive neurons resided outside the OVLT in the dorsal area and did not overlap with the Nax-positive population at all (Fig. 8D–F). Therefore, the possibility that Nax-positive glial cells envelop GABA neurons is low in the OVLT. These findings suggest that glial lamellate processes expressing Nax channels contact various neurochemical circuitries in the SFO and OVLT, encompassing GABAergic interneurons in the SFO.
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Nax channels populate perineuronal glial processes and thus appear to regulate neural activities, including that of GABAergic neurons, through glia–neuron communication in the sensory CVOs. Glial cells have long been considered to be inert partners of neurons in the central nervous system. It is becoming evident, however, that glial cells are intimately involved in neuronal signalling (Newman & Volterra, 2004). Our studies on Nax–/– mice demonstrated that the Nax channel exerts inhibitory influences on neuronal activities in the SFO and OVLT, as judged from Fos immunoreactivity during dehydration (Watanabe et al. 2000). There exist GABAergic neurons spontaneously firing in the SFO. We recently found that a hypertonic sodium solution enhanced the firing activity of the neurons of Nax+/+ but not Nax–/– mice using tissue slices (Fig. 9). Thus, the mechanism by which the sodium signal sensed by ‘inexcitable’ glial cells is transferred to neurons remains to be elucidated. The Nax channel function in the CVOs would provide a good system to study the molecular mechanisms for neuron–glia interactions.
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References
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Acknowledgements
This research was supported by grants from the Ministry of Education, Culture, Sports and Technology of Japan and Japan Science and Technology Agency (CREST). The author thanks Drs Takeshi Y. Hiyama and Eiji Watanabe for their contributions to this research and Ms Akiko Kodama for secretarial assistance.
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