1 Department of Physiology and Biophysics, Environmental and Hyperbaric Cell Biology Facility, Wright State University, Dayton, Ohio 45435; 2 Institut für Physiologie, Ruhr-Universität Bochum, D-44780 Bochum, Germany; 3 Department of Physiology and Biophysics, State University of New York at Stony Brook, Stony Brook 11794-8661; and 4 Department of Biology, Saint Lawrence University, Canton, New York 13617
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ABSTRACT |
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Gap junctions are composed of connexins, which are organized into intercellular channels that form transmembrane pathways between neurons (cell-cell coupling), and in some cases, neurons and glia, for exchange of ions and small molecules (metabolic coupling) and ionic current (electrical coupling). Cell-cell coupling via gap junctions has been identified in brain stem neurons that function in CO2/H+ chemoreception and respiratory rhythmogenesis; however, the exact roles of gap junctions in respiratory control are undetermined. Here we review the methods commonly used to study gap junctions in the mammalian brain stem under in vitro and in vivo conditions and briefly summarize the anatomical, pharmacological, and electrophysiological evidence to date supporting roles for cell-cell coupling in respiratory rhythmogenesis and central chemoreception. Specific research questions related to the role of gap junctions in respiratory control are suggested for future research.
brain stem; connexin; cell-cell coupling; central chemoreception; electrical coupling
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INTRODUCTION |
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GAP JUNCTIONS ARE INTERCELLULAR channels that form transmembrane pathways between neurons, and in some cases, neurons and glia, for exchange of ions (and ionic current) and small molecules. Thus gap junction channels provide the anatomical substrate for electrical coupling, allowing a neural network to function as an electrical ensemble, and for metabolic coupling, allowing the intercellular passage of small signaling molecules. Each cell contributes a hemichannel (connexon) to form a gap junction channel, and each connexon is composed of a hexameric assembly of structural proteins known as connexins (Cx). There are at least 15 Cx isoforms in rodents (8, 11, 15, 16, 21), and each Cx consists of four transmembrane domains, two extracellular loops, one cytoplasmic loop, and cytoplasmically located amino and carboxy termini (11, 35, 61, 62). The Cx isoforms incorporated into each gap junction channel define the physiological properties (i.e., permselectivity, gating, etc.) of the channel (10, 31); thus gap junction channels composed of different Cx exhibit different sensitivities to phosphorylation, transjunctional voltage, intracellular pH (pHi), and intracellular Ca2+ (27). In the mammalian brain, Cx26, Cx30, Cx32, Cx36, and Cx43 are the most abundantly expressed (7, 15, 20, 22, 32, 37, 38, 51, 60).
The first evidence for cell-cell coupling in a respiratory region of the brain stem was reported by Mazza et al. (34) when they observed dye coupling between adjoining hypoglossal motoneurons in slices prepared from neonatal rats <8 days old; presumably, Lucifer yellow iontophoresed into the recorded neuron was crossing into adjoining neurons through gap junctions. In 1997, further evidence for gap junctions in other respiratory centers of the brain stem was reported by Dean et al. (17) and Huang et al. (29) in neurons of the solitary complex (SC) [which comprises the nucleus tractus solitarius (NTS) and dorsal motor nucleus of vagus] and by Rekling and Feldman (47) in neurons of the nucleus ambiguus. Since that time, independent laboratories have reported additional evidence of cell-cell coupling in other respiratory centers, including the locus coeruleus (LC), ventrolateral medulla, medullary raphe, and phrenic motor nucleus (1, 5, 12, 18, 28, 41, 52). Much of this research was presented as a featured topic held during the Experimental Biology 2002 Meeting in New Orleans, LA. This well-attended session was sponsored by the Respiration Section of the American Physiological Society. The session provided an overview of the anatomical and electrophysiological evidence for gap junctions between neurons residing in respiratory control centers and included research using both whole animal and in vitro (brain slice, isolated brain stem-spinal cord) preparations. Several important issues were raised by the speakers as well as by the audience during the ensuing discussion periods regarding the function of gap junctions in respiratory control and the effects of changes in junctional conductance (Gj) on respiratory output and central chemosensitivity. These issues are summarized below.
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SUMMARY OF METHODS USED TO IDENTIFY GAP JUNCTIONS IN BRAIN STEM NEURONS |
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Gap junctions are detected using both direct and indirect electrophysiological and anatomical methods. Direct anatomical evidence for gap junctions includes immunohistochemical labeling of Cx proteins (12, 53, 54) and verification of gap junctions in thin section and freeze-fracture transmission electron micrographs (39, 46). Although there is extensive evidence for localization of Cx in respiratory centers of the neonatal and adult rat brain stem (12, 52-54), transmission electron microscopy evidence for the brain stem is lacking.
Direct electrophysiological evidence for gap junctions includes measurement of Gj or the electrical coupling coefficient (Kc) in pairs of neurons (14, 47, 49, 57). Measurement of Gj or Kc involves recording from two neurons simultaneously, under in vitro conditions, while making reciprocal current injections in an alternating fashion between neurons and observing the magnitude of coincident membrane potential (Vm) changes in each neuron. This experiment, however, is technically challenging and is often confounded by changes in nonjunctional conductances and the long electrotonic length between neurons in the brain slice and isolated brain stem-spinal cord (18). However, we reported preliminary findings, during the featured topic session, that in paired recording from LC neurons, cross-power spectral analysis of the Vm response to low-frequency (<1 Hz) sinuosoidal current injection revealed evidence of electrical coupling that was significantly reduced (lowered Kc) during exposure to the gap junction uncoupling agent carbenoxolone (D. Ballantyne, unpublished observations).
Alternatively, indirect measures of electrical coupling are frequently used to estimate changes in Gj between brain stem neurons. These include measurement of synchronized oscillations of Vm and/or action potential firing during simultaneous whole cell or perforated-patch recordings from pairs of neurons. Vm oscillations without action potentials are referred to as subthreshold rhythmical oscillations (SROs) and are typical features of neurons in the LC and inferior olive. Although their occurrence in an individual neuron does not denote electrical coupling per se, their synchronous occurrence within the LC network is dependent on electrical coupling (2, 5, 18, 41). In addition, single-electrode recordings of spontaneous electrotonic postsynaptic potentials in SC neurons (18, 29) and extracellular recordings of short latency depolarization potentials (4) in hypoglossal neurons (34) and phrenic neurons (28) are also used to estimate changes in electrical coupling.
Indirect measures of anatomical coupling include observation of dye coupling with Lucifer yellow and/or tracer coupling with biocytin or neurobiotin (13, 17, 29). Frequently, anatomical coupling is the first evidence for gap junctions in a region of the central nervous system (CNS). However, it is important that sources of staining artifacts that result in false positives are minimized or avoided. Likewise, negative results in an anatomical coupling study are not proof that gap junctions are absent (17, 18).
Pharmacological agents known to effectively uncouple gap junctions
(i.e., reduce or abolish ionic current flow and flow of dye between
cells) are also used to study how uncoupling alters the activity of
individual neurons, the neural drive for breathing (5, 18,
41), and expired minute ventilation (E)
(23). The effectiveness of these compounds as uncoupling
agents has been established in various neural and nonneural tissues
based on direct measurements of cell-cell coupling (18, 21, 52, 55, 57). Some of the uncoupling agents used to study cell-cell coupling in brain stem neurons include carbenoxolone, heptanol, 100%
CO2, and halothane (5, 18, 30, 41, 57).
However, many uncoupling agents also have additional effects on
nonjunctional conductances, and this, in addition to gap junction
uncoupling, may account for the loss of spontaneous activity and
disappearance of Vm oscillations that usually
occur during exposure to uncoupling agents (18).
Carbenoxolone, however, appears to be relatively "clean" as an
uncoupling agent because it does not abolish spontaneous firing and
does not have the nonspecific effects on membrane input resistance
observed with other uncoupling agents (18). Thus carbenoxolone has been used extensively to study the effects of gap
junction (i.e., electrical) uncoupling on respiratory control in the
awake rat (23) and in transverse medullary slice and isolated brain stem-spinal cord preparations of neonatal rodents (3, 5, 41).
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DISTRIBUTION OF GAP JUNCTIONS IN THE BRAIN STEM: CX EXPRESSION |
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Whole animal and in vitro studies indicate that central CO2 chemoreceptors are distributed throughout the brain stem, occurring in the SC (NTS, dorsal motor nucleus of vagus), ventrolateral medulla (including the retrofacial and retrotrapezoid nuclei), ventral respiratory group [including the pre-Bötzinger complex (pre-BötC)], caudal medullary raphe, and LC; see reviews by Solomon et al. (52, 53) and Nattie (40). Recently, Solomon and colleagues (53, 54) reported neuronal and astrocytic expression of Cx26 and neuronal expression of Cx32 in each of the putative CO2-chemosensitive brain stem regions described above as well as in the region containing the presumptive respiratory rhythm generator, the pre-BötC (25, 48, 50). In general, neuronal expression was characterized by intense polar or diffuse cytosolic, perinuclear (seen as a ring surrounding the nucleus), punctate, and dendritic process labeling, and astrocytic expression was characterized by starlike planar filament and punctate labeling. Furthermore, immunoblot analyses on microdissected tissue samples from these regions and immunohistochemical localization of Cx in each of these specific respiratory-related nuclei revealed regional and developmental differences in Cx expression in these regions [see Solomon et al. (53) for the precise patterns of Cx26 and Cx32 labeling observed (i.e., rostrocaudal regional differences, characteristics of neuronal and astrocytic labeling, developmental changes in Cx expression, etc.) in the putative CO2-chemosensitive brain stem regions]. Expression of Cx36 mRNA has also been reported in the NTS and pre-BötC in adult mice (42), and preliminary data, which were presented at the featured topic session, also demonstrated the presence of this neuron-specific Cx36 (15, 51) in each of the putative CO2-chemosensitive brain stem regions in both neonatal and adult rat (Solomon, unpublished observations).
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RESPIRATORY RHYTHMOGENESIS |
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As already stated and summarized elsewhere (52), Cx expression, electrical coupling, and anatomical coupling have been identified in motoneurons in respiratory-related motor nuclei (12). Both neuronal and astrocytic Cx expression have been reported in the hypoglossal and phrenic motor nuclei in both neonatal and adult rat (12). However, to date, electrical and anatomical coupling has only been demonstrated in these regions in embryonic and neonatal rats (28, 34). Although the exact function of cell-cell coupling in these respiratory-related motoneurons is unknown, a possible role may be to produce synchronized firing in respiratory-related motoneurons to coordinate muscular contractions (33, 34) and to synchronize descending respiratory drive to the diaphragm (28).
In isolated brain stem-spinal cord preparations in the neonatal rat, synchronized neural activity is indicative of cell-cell coupling, and initial results reveal that exposure to the gap junction uncoupling agent carbenoxolone produces a fivefold decrease in the electrical Kc of LC neurons and suppresses their SRO activity. It does not, however, suppress their respiratory-synchronous innervation or their capacity for spike discharge, which continues at a level set by the prevailing Vm. In addition, carbenoxolone does not exert any significant effect on integrated phrenic burst amplitude, but it markedly reduces phrenic burst (= respiratory) frequency (see also Ref. 9). The implication is that although the respiratory rhythm generator in this preparation is not dependent on intact gap junction coupling, such coupling does influence the length of the interval between each burst. This effect is not attributable to a loss of chemosensitivity because, in the presence of carbenoxolone, raising the CO2 concentration (2-10%) increases both phrenic burst frequency (i.e., the typical response to CO2 in this preparation) and the discharge frequency of LC neurons (i.e., the typical response of LC neurons to CO2).
In awake rats ranging from 6 to 8 wk old, unilateral focal perfusion of
carbenoxolone into the NTS, which uncouples gap junctions and reduces
Kc, decreases E by decreasing
tidal volume (Vt) but not respiratory frequency. The
inhibitory effect of NTS uncoupling on Vt, however,
disappeared in rats by 13 wk or older, suggesting a reduction in the
role of gap junctions in setting spontaneous breathing, at least in the
NTS (23). Importantly, focal acidification of the NTS
elicited similar increases in
E regardless of age. These findings suggest that although the respiratory response to acidic
stimuli is similar in young and old adults alike, the underlying neural
circuitry involving gap junctions in the NTS may undergo significant
developmental changes. It is possible that early in development,
electrical coupling may play an important role in chemosensory function
in some areas until individual neurons develop their full complement of
ion channels required to effectively operate as independent
chemosensors (i.e., they express pH-sensitive ion channels) (6,
26, 58, 59). In this way, the chemosensory output could be
maintained regardless of age, although the underlying neural
circuitry/connectivity may differ: electrical coupling may predominate
in young animals, and chemical synaptic transmission may predominate in
older animals. Consistent with this idea, Rao and colleagues
(45) found that synaptophysin (a marker for chemical synaptic transmission) staining increased threefold between birth and
postnatal day 9 and twofold between postnatal day
9 and postnatal day 70. Furthermore, Miller et
al. (36) showed a 1.3-fold increase in presynaptic
boutons/unit volume between postnatal day 30 and adulthood.
Together, these data suggest that chemical synaptogenesis develops over
a fairly long time course that extends up to at least 10 wk of age in
the rat, and this time course is consistent with a trade off from
electrical to chemical communication among neurons over the course of development.
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CENTRAL CO2/H+ CHEMORECEPTION |
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As stated above, Cx26, Cx32, and the recently identified Cx36 have been localized in all brain stem regions in neonatal and adult rats in which central CO2/H+ chemoreceptors are believed to reside (52, 53). Neurophysiological research to date, however, has only focused on two areas, the SC and LC. Dean et al. (18) and Huang et al. (29) found that SC neurons that were stimulated by hypercapnic acidosis were also anatomically and electrically coupled. In contrast, CO2-insensitive neurons in the SC rarely exhibited evidence of anatomical and electrical coupling. Likewise, LC neurons exhibited a high incidence of CO2 chemosensitivity (5, 26, 41) and cell-cell coupling (2, 18, 41). The occurrence of gap junctions between adjoining chemosensitive neurons suggests that either electrical coupling or metabolic coupling may be involved in central chemoreception of CO2 and H+ and cardiorespiratory control. As pointed out elsewhere (18), this hypothesis is intriguing because intracellular acidification, which has been proposed as the proximate stimulus for the central chemoreceptors (44), is also reported to uncouple gap junctions in many types of networks, either directly via increased [H+] (56) or indirectly via a pHi-dependent process (43). This raises the question, How sensitive is Gj in chemosensitive neurons to CO2-induced intracellular acidification?
The evidence to date suggests that Gj between chemosensitive neurons is pHi insensitive over a physiological range of hypercapnic acidosis. For example, in SC neurons, spontaneous electronic postsynaptic potentials were maintained during exposure to 15% CO2 with no significant change in their amplitude. Electrical uncoupling would have resulted in either decreased amplitude of electrotonic postsynaptic potentials or loss of any measurable postsynaptic activity (18, 29). Likewise, synchronous activity in pairs of LC neurons was retained during exposure to a broad range of hypercapnic acidosis (5, 18, 19). Measurements of pHi in pontine slices indicate that synchronized activity in the LC was retained in 93% of CO2 tests in cell pairs as pHi decreased from 7.24 to 6.96. Conversely, synchronized activity was retained in only 33% of neurons at pHi <6.96 (18, 19). At the lowest levels of intracellular acidosis, synchronized activity was either disrupted or abolished, suggesting that gap junctions were uncoupled and neuronal excitability was reduced. In addition, severe levels of hypercapnic acidosis also appear to increase nonjunctional conductance and reduce overall excitability (18, 19).
In addition, as stated above, cell-cell coupling does not appear to be required for expression of CO2 chemosensitivity, per se, in the single cell, integrated network, or intact animal (3, 23, 41). It may be that cell-cell coupling provides an anatomical site for modulating excitability of the chemoreceptor network to stimuli that modulate central chemosensitivity. Changes in the strength of cell-cell coupling (Gj) within a chemosensitive area could influence the sensitivity or gain of the entire chemoreceptor network to stimuli that alter central chemosensitivity. In this regard, it will be important to determine how changes in Gj affect local circuit processing in chemosensitive areas and the response to chemical synaptic afferents. It is also important to recognize that developmental changes in the strength or location of gap junctions may have highly significant effects in relation to their synchronizing action, e.g., the loss of synchronization among adult LC neurons as spike frequency is increased does not occur in the neonate, a difference that may be partly due to a redistribution of gap junctions (1). This suggests that in a coupled network of chemosensitive neurons, e.g., in the LC and SC, CO2-dependent changes in spike frequency may also result in a shift between synchronized and nonsynchronized states of the network.
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WHERE DO WE GO NEXT? |
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The neurobiology of gap junctions in central chemoreception and respiratory control is a relatively new area of respiratory neurophysiology. Investigators working in this new field are collaborating and using a broad array of techniques to study cell-cell coupling in brain stem respiratory centers, which includes immunohistochemistry, anatomical coupling, electrophysiology, and whole animal neurophysiology. Future research needs to continue using this multifaceted and collaborative approach to identify how changes in Cx expression and strength of cell-cell coupling affects respiratory drive. Many unresolved issues were raised at this featured topic this year. For example, What is the function of Cx proteins residing in the cytosol? Although cytosolic expression of Cx is suggested to represent the trafficking (i.e., synthesis, assembly, and degradation of gap junction channels) and intracellular storage of gap junction proteins between the Golgi apparatus and the plasma membrane (24, 63), the use of transmission electron microscopy and confocal microscopy will help to further resolve the distribution of Cx in the neuron and help elucidate the precise role of intracellular Cx. How does the strength of cell-cell coupling within brain stem centers alter the effects of impinging chemical synaptic inputs? Moreover, how does a physiological change in pHi, as well as other factors that modulate central chemosensitivity and/or respiratory control, affect Gj between respiratory-related neurons? These have been difficult questions to answer to date for reasons cited above. However, the challenge of directly measuring Gj in the brain slice and the isolated brain stem-spinal cord preparation has begun to be solved using slow current oscillations and cross-power spectral analysis (D. Ballantyne, unpublished observations). It is possible that this approach may reveal subtle changes in Gj during intracellular acidosis, which were previously undetected using indirect measures of electrical coupling. What is the extent of junctional coupling in various respiratory areas, taking into account the limitations of the methods used to identify cell-cell coupling in in vitro tissue preparations of the CNS (18)? Moreover, how do coupled networks in different respiratory areas of the brain stem interact with each other, if at all?
The challenge of answering these questions is that it is likely that there is no one simple answer. Cell-cell coupling is manifested differently in the regions that have been studied. For example, chemosensitive neurons in the SC exhibit electrotonic postsynaptic potentials and a high incidence of dye coupling, whereas neurons in the LC exhibit synchronized SROs and a low incidence of dye coupling. However, research on the role of gap junctions in respiratory control will provide important insights into how respiratory networks develop, function, and adapt to environmental stimuli.
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ACKNOWLEDGEMENTS |
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This work was supported by National Heart, Lung, and Blood Institute Grants R01-HL-63175 and R01-HL-16022 (to I. C. Solomon) and R01-HL-56683 (to J. B. Dean); National Science Foundation Grant IBN-9810809 (to J. S. Erlichman); Office of Naval Research Grant N000140110179 (to J. B. Dean); Merck-American Academy for the Advancement of Science and the Phelps Foundation (to J. S. Erlichman); and Deutsche Forschungsgemeinschaft (Sche 46/12-4) (to D. Ballantyne).
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FOOTNOTES |
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Address for reprint requests and other correspondence: J. B. Dean, Rm. 160 Bio. Sci. Bldg., Dept. of Physiology and Biophysics, 3640 Col. Glenn Hwy., Wright State Univ., Dayton, OH 45435 (E-mail: jay.dean{at}wright.edu).
10.1152/ajplung.00142.2002
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