Solitary Complex Research Articles

Further evidence of redox modulation of neurons in a CO2‐chemosensitive area: normobaric hyperoxia (95%O2) stimulates CO2‐chemosensitive and –insensitive neurons in the solitary complex (SC)

We previously reported that CO2‐excited neurons in SC, in medullary slices maintained in 95%O2, are stimulated by chemical oxidants and hyperbaric O2 (JAP 95:910‐921, 2003; AJP 286:C940–951, 2004). Here we test the hypothesis that SC neurons, maintained in 40%O2 rather than 95%O2 (to reduce oxidative stress), are viable and stimulated by normobaric hyperoxia (95%O2). Slices (P1–21) were harvested in chilled ACSF gassed with 95%O2 and immediately transferred to ACSF (22–24°C) equilibrated with 40%O2‐5%CO2 in N2. Whole cell recordings (35–37°C) were established in 40%O2 (n=44) and tested using hyperoxia (40→95%O2) and hypercapnia (5→10 or 15%CO2). Hyperoxia stimulated 8/44 neurons and hypercapnia stimulated 5/44 neurons; of these 4/8 were stimulated by both O2 and CO2. Hyperoxia usually increased firing rate and decreased input resistance whereas hypercapnia increased firing rate and input resistance. Most neurons tested were insensitive to hyperoxia and hypercapnia (31/44). Compared to previous slice studies that used 95%O2 control, there is a smaller proportion of CO2‐excited SC neurons in slices maintained in 40%O2. These data indicate that medullary slices are viable in 40%O2 and that hyperoxic stress stimulates SC neurons, including CO2‐excited neurons. These data also suggest that oxidative stimuli increases the incidence of CO2‐chemosensitivity in SC neurons (ONR N000140110179, NIH R01HL56683)

Oxygen‐induced superoxide production in solitary complex neurons in rat medullary slices

We previously reported that the rate of superoxide (•O2−) production in CA1 neurons in hippocampal slices decreases as O2 is decreased from 95% to 60, 40 or 20% (J. Neurophysiol. 98:1030‐1041, 2007). Here we report the effects of continuous exposure to O2 (95, 40 & 20%) on •O2− production in solitary complex (SC) neurons in medullary slices (400μm, weaned rats). The rate of intracellular •O2− production was measured using the fluorogenic probe, DHE (2.5μM), added to the superfusate (35–37°C). Fluorescence imaging initially classified cells at the start of the experiment as dim versus bright. Dim SC neurons incubated in 95%O2 had a similar rate of •O2− production (increasing fluorescence) over time as that reported previously for CA1 hippocampal neurons. Continuous exposure of dim cells to 20 or 40%O2, however, revealed slightly lower but similar rates of •O2− production that were significantly greater than that reported for CA1 neurons. SC neurons initially identified as bright cells increased their rate of fluorescence during the first 60–90 minutes but thereafter fluorescence reached a plateau indicative of a decreased rate of •O2− production. In bright SC cells, 95% and 20%O2 caused the lowest and highest rate of •O2− production, respectively. We conclude the following: 1) dim SC neurons have a higher capacity for •O2− production during exposure to 20–40%O2 than CA1 hippocampal neurons. 2) Neurons with initially bright DHE fluorescence may be damaged from slicing and lose their •O2− generating mechanisms rapidly, especially in the presence of 95%O2 (ONR N000140110179, NIH R01HL56683).

Central CO2chemoreception in cardiorespiratory control

Central CO₂chemoreception in cardiorespiratory control

Hypercapnia causes cellular oxidation and nitrosation in addition to acidosis: implications for CO2 chemoreceptor function and dysfunction.

Cellular mechanisms of CO2 chemoreception are discussed and debated in terms of the stimuli produced during hypercapnic acidosis and their molecular targets: protons generated by the hydration of CO2 and dissociation of carbonic acid, which target membrane-bound proteins and lipids in brain stem neurons. The CO2 hydration reaction, however, is not the only reaction that CO2 undergoes that generates molecules capable of modifying proteins and lipids. Molecular CO2 also reacts with peroxynitrite (ONOO-), a reactive nitrogen species (RNS), which is produced from nitric oxide (*NO) and superoxide (*O2-). The CO2/ONOO- reaction, in turn, produces additional nitrosative and oxidative reactive intermediates. Furthermore, protons facilitate additional redox reactions that generate other reactive oxygen species (ROS). ROS/RNS generated by these redox reactions may act as additional stimuli of CO2 chemoreceptors since neurons in chemosensitive areas produce both *NO and *O2- and, therefore, ONOO-. Perturbing *NO, *O2-, and ONOO- activities in chemosensitive areas modulates cardiorespiration. Moreover, neurons in at least one chemosensitive area, the solitary complex, are stimulated by cellular oxidation. Together, these data raise the following two questions: 1) do pH and ROS/RNS work in tandem to stimulate CO2 chemoreceptors during hypercapnic acidosis; and 2) does nitrosative stress and oxidative stress contribute to CO2 chemoreceptor dysfunction? To begin considering these two issues and their implications for central chemoreception, this minireview has the following three goals: 1) summarize the nitrosative and oxidative reactions that occur during hypercapnic acidosis and isocapnic acidosis; 2) review the evidence that redox signaling occurs in chemosensitive areas; and 3) review the evidence that neurons in the solitary complex are stimulated by cellular oxidation.

Chronic hypoxia suppresses the CO2 response of solitary complex (SC) neurons from rats

We studied the effect of chronic hypobaric hypoxia (CHx; 10-11% O(2)) on the response to hypercapnia (15% CO(2)) of individual solitary complex (SC) neurons from adult rats. We simultaneously measured the intracellular pH and firing rate responses to hypercapnia of SC neurons in superfused medullary slices from control and CHx-adapted adult rats using the blind whole cell patch clamp technique and fluorescence imaging microscopy. We found that CHx caused the percentage of SC neurons inhibited by hypercapnia to significantly increase from about 10% up to about 30%, but did not significantly alter the percentage of SC neurons activated by hypercapnia (50% in control vs. 35% in CHx). Further, the magnitudes of the responses of SC neurons from control rats (chemosensitivity index for activated neurons of 166+/-11% and for inhibited neurons of 45+/-15%) were the same in SC neurons from CHx-adapted rats. This plasticity induced in chemosensitive SC neurons by CHx appears to involve intrinsic changes in neuronal properties since they were the same in synaptic blockade medium.

Effects of a lower level of control O2, hyperoxia and hypercapnia on solitary complex neurons in medullary tissue slices

Studies of neuronal O2‐/CO2‐chemosensitivity in the brainstem often employ tissue slices maintained in medium equilibrated with 95%O2, which we now know produces hyperoxia, increased production of reactive species, and oxidative stress. Accordingly, the objectives of this study were to determine if solitary complex (SC) neurons in medullary slices, incubated in 40%O2, exhibit spontaneous, stable activity and are responsive to hyperoxia and hypercapnia. Medullary slices (300‐400µm, P3‐P21) were harvested in 95%O2 + 5%CO2 (2‐4°C) and immediately transferred to 40%O2 + 5%CO2 (22‐24°C). Whole‐cell recordings (~37°C) of 69 neurons show a mean resting membrane potential of ‐42mV with spontaneous and evoked action potentials that overshoot 0mV. Whole cell recordings were made within 2‐7.5hr post‐slicing. To date, 25 neurons were tested using hyperoxia (95% O2 + 5% CO2), hypercapnia (40% O2 + 10%CO2) and hyperoxic hypercapnia (90%O2 + 10%CO2). The majority of neurons (13) were insensitive to hyperoxia/hypercapnia. Eight neurons were stimulated by hyperoxia and five by hypercapnia. Of these, four were stimulated by hyperoxic hypercapnia. Two neurons were inhibited by hypercapnia and one was inhibited by hyperoxia. We conclude that medullary slices can be maintained in 40%O2 for many hours for studies of CO2/O2‐chemosensitivity at normobaric pressure. (ONR N000140710890, NIH R01 HL 56683‐09)

Commentaries on Viewpoint: Central chemoreception is a complex system function that involves multiple brain stem sites

to the editor: Amniote vertebrates adjust the acid-base status of the blood through ventilatory alterations of PaCO2 and, to some degree, by renal modulation of bicarbonate concentration. Receptor systems for the ventilatory acid-base regulation has been studied mainly in mammals, in which the

Characterization of the chemosensitive response of individual solitary complex neurons from adult rats

We studied the CO(2)/H(+)-chemosensitive responses of individual solitary complex (SC) neurons from adult rats by simultaneously measuring the intracellular pH (pH(i)) and electrical responses to hypercapnic acidosis (HA). SC neurons were recorded using the blind whole cell patch-clamp technique and loading the soma with the pH-sensitive dye pyranine through the patch pipette. We found that SC neurons from adult rats have a lower steady-state pH(i) than SC neurons from neonatal rats. In the presence of chemical and electrical synaptic blockade, adult SC neurons have firing rate responses to HA (percentage of neurons activated or inhibited and the magnitude of response as determined by the chemosensitivity index) that are similar to SC neurons from neonatal rats. They also have a typical response to isohydric hypercapnia, including decreased DeltapH(i), followed by pH(i) recovery, and increased firing rate. Thus, the chemosensitive response of SC neurons from adults is similar to the chemosensitive response of SC neurons from neonatal rats. Because our findings for adults are similar to previously reported values for neurons from neonatal rats, we conclude that intrinsic chemosensitivity is established early in development for SC neurons and is maintained throughout adulthood.

Estrogen receptor-α immunoreactive neurons in the brainstem and spinal cord of the female rhesus monkey: Species-specific characteristics

The distribution pattern of estrogen receptors in the rodent CNS has been reported extensively, but mapping of estrogen receptors in primates is incomplete. In this study we describe the distribution of estrogen receptor alpha immunoreactive (ER-α IR) neurons in the brainstem and spinal cord of the rhesus monkey.In the midbrain, ER-α IR neurons were located in the periaqueductal gray, especially the caudal ventrolateral part, the adjacent tegmentum, peripeduncular nucleus, and pretectal nucleus. A few ER-α IR neurons were found in the lateral parabrachial nucleus, lateral pontine tegmentum, and pontine gray medial to the locus coeruleus. At caudal medullary levels, ER-α IR neurons were present in the commissural nucleus of the solitary complex and the caudal spinal trigeminal nucleus. The remaining regions of the brainstem were devoid of ER-α IR neurons. Spinal ER-α IR neurons were found in laminae I–V, and area X, and were most numerous in lower lumbar and sacral segments. The lateral collateral pathway and dorsal commissural nuclei of the sacral cord and the thoracic intermediolateral cell column also contained ER-α IR neurons. Estrogen treatment did not result in any differences in the distribution pattern of ER-α IR neurons.The results indicate that ER-α IR neurons in the primate brainstem and spinal cord are concentrated mainly in regions involved in sensory and autonomic processing. Compared with rodent species, the regional distribution of ER-α IR neurons is less widespread, and ER-α IR neurons in regions such as the spinal dorsal horn and caudal spinal trigeminal nucleus appear to be less abundant. These distinctions suggest a modest role of ER-α in estrogen-mediated actions on primate brainstem and spinal systems. These differences may contribute to variations in behavioral effects of estrogen between primate and rodent species.

Target cell-specific difference in the release probability defines synaptic filtering in the solitary complex in the rat

Target cell-specific difference in the release probability defines synaptic filtering in the solitary complex in the rat

Short-term plasticity defines postsynaptic excitation pattern in the solitary complex neurons of the rat

Short-term plasticity defines postsynaptic excitation pattern in the solitary complex neurons of the rat

First localization and biochemical identification of chromogranin B- and secretoneurin-like immunoreactivity in the fetal human vagal/nucleus solitary complex

The human vagal/nucleus solitary complex is a primary visceral relay station and an integrative brain stem area which displays a high density of chromogranin B- and secretoneurin-like immunoreactivity. In this study, we localized and biochemically identified these proteins during prenatal development. At prenatal week 11, 15, 20 and 37, we performed a chromatographic analysis to identify the molecular forms of PE-11, a peptide within the chromogranin B sequence, and secretoneurin, a peptide within secretogranin II. Their localization was studied with immunocytochemistry, and was compared to that of substance P which is well established as a functional neuropeptide in the vagal/nucleus solitary complex. At prenatal week 11, chromogranin B-, secretoneurin- and substance P-like immunoreactivities were detected consisting of varicosities, varicose fibers and single cells. At the same time, PE-11 and secretoneurin appeared as a single peak in chromatographic analysis. Prohormone convertases PC1- and PC2-like immunoreactivities were also present at week 11. In general, the density for each peptide increased during later fetal stages with the highest density at week 37. These results demonstrate that each chromogranin peptide is expressed during human fetal life in neurons of the vagal/nucleus solitary complex indicating that these peptides could be important during prenatal development.

Distinct presynaptic mechanisms underlie firing frequency-dependent modulation of synaptic transmission in the solitary complex

Distinct presynaptic mechanisms underlie firing frequency-dependent modulation of synaptic transmission in the solitary complex

Hyperoxia, reactive oxygen species, and hyperventilation: oxygen sensitivity of brain stem neurons.

Hyperoxia is a popular model of oxidative stress. However, hyperoxic gas mixtures are routinely used for chemical denervation of peripheral O2 receptors in in vivo studies of respiratory control. The underlying assumption whenever using hyperoxia is that there are no direct effects of molecular O2 and reactive O2 species (ROS) on brain stem function. In addition, control superfusates used routinely for in vitro studies of neurons in brain slices are, in fact, hyperoxic. Again, the assumption is that there are no direct effects of O2 and ROS on neuronal activity. Research contradicts this assumption by demonstrating that O2 has central effects on the brain stem respiratory centers and several effects on neurons in respiratory control areas; these need to be considered whenever hyperoxia is used. This mini-review summarizes the long-recognized, but seldom acknowledged, paradox of respiratory control known as hyperoxic hyperventilation. Several proposed mechanisms are discussed, including the recent hypothesis that hyperoxic hyperventilation is initiated by increased production of ROS during hyperoxia, which directly stimulates central CO2 chemoreceptors in the solitary complex. Hyperoxic hyperventilation may provide clues into the fundamental role of redox signaling and ROS in central control of breathing; moreover, oxidative stress may play a role in respiratory control dysfunction. The practical implications of brain stem O2 and ROS sensitivity are also considered relative to the present uses of hyperoxia in respiratory control research in humans, animals, and brain stem tissues. Recommendations for future research are also proposed.

Oxidative stress decreases pHi and Na(+)/H(+) exchange and increases excitability of solitary complex neurons from rat brain slices.

Putative chemoreceptors in the solitary complex (SC) are sensitive to hypercapnia and oxidative stress. We tested the hypothesis that oxidative stress stimulates SC neurons by a mechanism independent of intracellular pH (pH(i)). pH(i) was measured by using ratiometric fluorescence imaging microscopy, utilizing either the pH-sensitive fluorescent dye BCECF or, during whole cell recordings, pyranine in SC neurons in brain stem slices from rat pups. Oxidative stress decreased pH(i) in 270 of 436 (62%) SC neurons tested. Chloramine-T (CT), N-chlorosuccinimide (NCS), dihydroxyfumaric acid, and H(2)O(2) decreased pH(i) by 0.19 +/- 0.007, 0.20 +/- 0.015, 0.15 +/- 0.013, and 0.08 +/- 0.002 pH unit, respectively. Hypercapnia decreased pH(i) by 0.26 +/- 0.006 pH unit (n = 95). The combination of hypercapnia and CT or NCS had an additive effect on pH(i), causing a 0.42 +/- 0.03 (n = 21) pH unit acidification. CT slowed pH(i) recovery mediated by Na(+)/H(+) exchange (NHE) from NH(4)Cl-induced acidification by 53% (n = 20) in CO(2)/HCO(3)(-)-buffered medium and by 58% (n = 10) in HEPES-buffered medium. CT increased firing rate in 14 of 16 SC neurons, and there was no difference in the firing rate response to CT with or without a corresponding change in pH(i). These results indicate that oxidative stress 1). decreases pH(i) in some SC neurons, 2). together with hypercapnia has an additive effect on pH(i), 3). partially inhibits NHE, and 4) directly affects excitability of CO(2)/H(+)-chemosensitive SC neurons independently of pH(i) changes. These findings suggest that oxidative stress acidifies SC neurons in part by inhibiting NHE, and this acidification may contribute ultimately to respiratory control dysfunction.

The neuronal circuit of augmenting effects on intrinsic laryngeal muscle activities induced by nasal air-jet stimulation in decerebrate cats

We previously demonstrated that during nasal air-jet stimulation, both the activities of intrinsic laryngeal adductor and abductor muscles persistently increase, whereas the respiratory cycle prolongs and the activity of diaphragm decreases [Am. J. Rhinol. 9 (1995) 203–208; Neurosci. Res. 31 (1998) 137–146]. The purpose of this study was to clarify the neuronal circuit underlying the augmentation of intrinsic laryngeal muscles evoked by nasal air-jet stimulation. The immunohistologic analysis of Fos-expression was reported to determine the distribution of activated neurons in cat brainstem evoked by sneeze-inducing air puff stimulation of the nasal mucosa [Brain Res. 687 (1995) 143–154]. In sneezing cats, immunoreactivity was evoked in projection areas of the ethmoidal afferents, e.g. the subnuclei caudalis, interpolaris and in interstitial islands of the trigeminal sensory complex. Immunoreactivity was also enhanced in the solitary complex, the nucleus retroambiguus, the pontine parabrachial area and the lateral aspect of the parvocellular reticular formation [Brain Res. 687 (1995) 143–154]. In the present study, we focussed on the parvocellular reticular nucleus (PRN) as a relay of the neural circuit contributed to the augmentation of intrinsic laryngeal muscles evoked by nasal air-jet stimulation. We recorded the neuronal behavior of PRN during the nasal air-jet stimulation in precollicular-postmammillary decerebrate cats. As the results, 24% (17/71) of recorded neurons which were activated orthodromically by the electrical stimulation to anterior ethmoidal nerve, increased their firing rates in response to the nasal air-jet stimulation. Furthermore, spike-triggered averaging method revealed that four of these 17 PRN neurons activated intrinsic laryngeal muscles, suggesting that such neurons have excitatory projections to the intrinsic laryngeal muscle motoneurons in the nucleus ambiguus. These results suggest that the some of PRN neuron play a role in augmentation of the intrinsic laryngeal muscles activities during nasal air-jet stimulation.

Pressure (< or=4 ATA) increases membrane conductance and firing rate in the rat solitary complex.

Neuronal sensitivity to pressure, barosensitivity, is illustrated by high-pressure nervous syndrome, which manifests as increased central nervous system excitability when heliox or trimix is breathed at >15 atmospheres absolute (ATA). We have tested the hypothesis that smaller levels of pressure (<or=4 ATA) also increase neuronal excitability. The effect of hyperbaric helium, which mimics increased hydrostatic pressure, was determined on putative CO2/H+-chemoreceptor neurons in the solitary complex in rat brain stem slices by intracellular recording. Pressure stimulated firing rate in 31% of neurons (barosensitivity) and decreased input resistance. Barosensitivity was retained during synaptic blockade and was unaffected by antioxidants. Barosensitivity was distributed among CO2/H+-chemosensitive and -insensitive neurons; in CO2/H+-chemosensitive neurons, pressure did not significantly reduce neuronal chemosensitivity. We conclude that moderate pressure stimulates certain solitary complex neurons by a mechanism that possibly involves an increased cation conductance, but that does not involve free radicals. Neuronal barosensitivity to <or=4 ATA may represent a physiological adaptive response to increased pressure or a pathophysiological response that is the early manifestation of high-pressure nervous syndrome.

Hyperbaric oxygen and chemical oxidants stimulate CO2/H+-sensitive neurons in rat brain stem slices.

Hyperoxia, a model of oxidative stress, can disrupt brain stem function, presumably by an increase in O2 free radicals. Breathing hyperbaric oxygen (HBO2) initially causes hyperoxic hyperventilation, whereas extended exposure to HBO2 disrupts cardiorespiratory control. Presently, it is unknown how hyperoxia affects brain stem neurons. We have tested the hypothesis that hyperoxia increases excitability of neurons of the solitary complex neurons, which is an important region for cardiorespiratory control and central CO2/H+ chemoreception. Intracellular recordings were made in rat medullary slices during exposure to 2-3 atm of HBO2, HBO2 plus antioxidant (Trolox C), and chemical oxidants (N-chlorosuccinimide, chloramine-T). HBO2 increased input resistance and stimulated firing rate in 38% of neurons; both effects of HBO2 were blocked by antioxidant and mimicked by chemical oxidants. Hypercapnia stimulated 32 of 60 (53%) neurons. Remarkably, these CO2/H+-chemosensitive neurons were preferentially sensitive to HBO2; 90% of neurons sensitive to HBO2 and/or chemical oxidants were also CO2/H+ chemosensitive. Conversely, only 19% of HBO2-insensitive neurons were CO2/H+ chemosensitive. We conclude that hyperoxia decreases membrane conductance and stimulates firing of putative central CO2/H+-chemoreceptor neurons by an O2 free radical mechanism. These findings may explain why hyperoxia, paradoxically, stimulates ventilation.

Hyperbaric oxygen depolarizes solitary complex neurons in tissue slices of rat medulla oblongata.

Hyperbaric oxygen (HBO2) at approximately 3 atmospheres absolute (ATA) pressure is toxic to the mammalian CNS due to excessive O2 free radical production. No study has ever determined the effects of < or = 3 ATA of O2 on the membrane potential and firing rate of neurons in the mammalian brainstem. Likewise, no study has ever determined the effects of < or = 3 ATA pressure per se on brainstem neurons. Accordingly, we initiated intracellular recordings at 1 ATA in solitary complex neurons in slices (300 microns) of rat caudal medulla oblongata that were maintained inside a 72 liter hyperbaric chamber. Helium, which is inert and without narcotic effect at moderate levels of hyperbaria, was used to hydrostatically compress the submerged brain slice to determine the effects of pressure per se. Tissue oxygen tension and extracellular pH were also measured during exposure to hyperbaric gases. Six of 19 neurons were affected by hyperbaric helium; 5 cells were depolarized and 1 cell was hyperpolarized. Input resistance (Rin) either increased (n = 1) or decreased (n = 3). When control perfusate (0.95 ATA O2) was switched to perfusate saturated with 98% O2 (balance CO2, pH = 7.3-7.4, pO2 = 2.5-3.4 ATA; 2-18 minutes of exposure) in a separate pressure vessel, 8 of 13 neurons were depolarized and 5 neurons were insensitive. In the 8 O2-responsive neurons, Rin either increased (n = 5), decreased (n = 2) or was unchanged (n = 1). Three of 8 neurons depolarized by HBO2 were also depolarized by hyperbaric helium, usually with an additional change in Rin. We conclude that hydrostatic (helium) pressure and HBO2 independently increase excitability in certain solitary complex neurons. We hypothesize that these responses contribute, in part, to neural events that either precede or occur during CNS O2 toxicity.

Cell–cell coupling in CO 2/H +-excited neurons in brainstem slices

The indirect and direct electrical and anatomical evidence for the hypothesis that central chemoreceptor neurons in the dorsal brainstem (solitary complex, SC; locus coeruleus, LC) are coupled by gap junctions, as reported primarily in rat brainstem slices, and the methods used to study gap junctions in brain slices, are critiqued and reviewed. Gap junctions allow intercellular communication that could be important in either electrical coupling (intercellular flow of ionic current), metabolic coupling (intercellular flow of signaling molecules), or both, ultimately influencing excitability within the SC and LC during respiratory acidosis. Gap junctions may also provide a mechanism for modulating neuronal activity in the network under conditions that lead to increased or decreased central respiratory chemosensitivity. Indirect measures of electrical coupling suggest that junctional conductance between chemosensitive neurons is relatively insensitive to a broad range of intracellular pH (pH i), ranging from pH i ≈7.49 to ≈6.71 at 35–37 °C. In contrast, further reductions in pH i, down through pH i ≈6.67, abolish indirect measures of electrical coupling.