Focal Acidification Research Articles

Focal microdialysis of CO2 in the perifornical-hypothalamic area increases ventilation during wakefulness but not NREM sleep

We investigated whether the perifornical-lateral hypothalamic area (PF-LHA), where the orexin neurons reside, is a central chemoreceptor site by microdialysis of artificial cerebrospinal fluid (aCSF) equilibrated with 25% CO2 into PF-LHA in conscious rats. This treatment is known to produce a focal tissue acidification like that associated with a 6–7mm Hg increase in arterial PCO2. Such focal acidification in the PF-LHA significantly increased ventilation up to 15% compared with microdialysis of normal aCSF equilibrated with 5% CO2 only in wakefulness but not in sleep in both the dark (P=0.004) and light (P<0.001) phases of the diurnal cycle. This response was predominantly due to a significant increase in respiratory frequency (11%, P<0.001). There were no significant effects on ventilation in the group with probes misplaced outside the PF-LHA. These results suggest that PF-LHA functions as a central chemoreceptor site in the central nervous system in a vigilant state dependent manner with predominant effects in wakefulness.

Focal acidification of the caudal ventrolateral medulla (Loeschcke's area) increases ventilation during wakefulness but not in sleep

The classic central chemoreceptors were originally described as the regions bilaterally located on the surface of the rostral and caudal ventrolateral medulla (rVLM and cVLM). The cVLM, just lateral to the pyramidal tract between the roots of the hypoglossal nerve, also known as Loeschcke's area, was excited by focal application of H+ and subsequently has not received much of attention in the past decade. Salgueiro et al. recently showed that some of the neurons in the cVLM increased firing rate in response to focal acidification in anaesthetized rats (2003). Here we hypothesize that in conscious rat, focal acidification of the cVLM region would increase ventilation in a sleep‐awake state dependent manner. In unanesthetized rats, normal (5%) or high (25%) CO2 equilibrated aCSF was unilaterally dialyzed into the cVLM region and ventilation was recorded using a whole body plethysmograph. In the responsive group (n=8), focal acidification with high CO2 significantly increased ventilation by 17% in wakefulness due to both VT and frequency but not in sleep comparing with the baseline. In the non‐responsive group (n=7), focal acidification did not affect VE, f and VT either in wakefulness or sleep. The focal acidification data suggest that the cVLM, the area that is partially overlapping Loeschcke's area, participates in the central chemoreception in an arousal state dependent manner. (HL‐28066)

Central chemoreception is a complex system function that involves multiple brain stem sites

central chemoreception refers to detection of CO2/pH within the brain and the subsequent reflex effects on breathing. It involves multiple sites within the hindbrain ([9][1], [12][2], [19][3]) as focal acidification in vivo uniquely at these sites stimulates breathing, indicating detection and

Focal CO2 dialysis in raphe obscurus does not stimulate ventilation but enhances the response to focal CO2 dialysis in the retrotrapezoid nucleus.

Simultaneous inhibition of the retrotrapezoid nucleus (RTN) and raphe obscurus (ROb) decreased the systemic CO(2) response by 51%, an effect greater than inhibition of RTN (-24%) or ROb (0%) alone, suggesting that ROb modulates chemoreception by interaction with the RTN (19). We investigated this interaction further by simultaneous dialysis of artificial cerebrospinal fluid equilibrated with 25% CO(2) in two probes located in or adjacent to the RTN and ROb in conscious adult male rats. Ventilation was measured in a whole body plethysmograph at 30 degrees C. There were four groups (n = 5): 1) probes correctly placed in both RTN and ROb (RTN-ROb); 2) one probe correctly placed in RTN and one incorrectly placed in areas adjacent to ROb (RTN-peri-ROb); 3) one probe correctly placed in ROb and one probe incorrectly placed in areas adjacent to RTN (peri-RTN-ROb); and 4) neither probe correctly placed (peri-RTN-peri-ROb). Focal simultaneous acidification of RTN-ROb significantly increased ventilation (Ve) up to 22% compared with baseline, with significant increases in both breathing frequency and tidal volume. Focal acidification of RTN-peri-ROb increased Ve significantly by up to 15% compared with baseline. Focal acidification of ROb and peri-RTN had no significant effect. The simultaneous acidification of regions just outside the RTN and ROb actually decreased Ve by up to 11%. These results support a modulatory role for the ROb with respect to central chemoreception at the RTN.

RAPHE OBSCURUS (ROb) MODULATES THE VENTILATORY RESPONSE TO FOCAL ACIDIFICATION OF THE RETROTRAPEZOID NUCLEUS (RTN)

Recent data suggest that the caudal medullary raphe (MR) does not contribute to chemoreception directly but modulates breathing by interaction with the RTN (Li et al., J Physiol 2006). To investigate this hypothesis, we dialyzed artificial cerebrospinal fluid equilibrated with 25% CO2 into RTN and ROb simultaneously in male rats in a whole body plethysmograph at a temperature within the rat thermoneutral zone (30°C). We compared data from animals with both probes correctly placed to data from rats with both probes misplaced. Simultaneous acidification of RTN and ROb increased VE up to 23%, compared with the baseline and up to 28% compared to the control group, since focal acidification in the control group decreased VE. The responses were similar in wakefulness and sleep. For single site dialysis experiments, both probes received 25% CO2 dialysis, but just one of the probes was correctly placed. The 25% CO2 dialysis into RTN caused a small increase in VE (∼8%; n=05), and this effect was due to an increased f. The focal acidification of the ROb had no effect on VE (N=04). Although there was no response to CO2 dialysis in the ROb, the simultaneous dialysis into the RTN and ROb potentiated the effect of the focal acidification of RTN alone. These results indicate a modulatory role of caudal MR with respect to central chemoreception at the RTN. (Supported by FAPESP and R37 HL 28066)

Raphe neuronal circuits and responses to central and peripheral chemoreceptor stimulation

Current models propose that raphe neurons are elements of both central and peripheral chemoreceptor reflex circuits. Focal acidification of the brainstem midline raphe can stimulate breathing and cultured raphe neurons have chemosensitive properties. However, little is known about the responses of individual raphe neurons in vivo and the network that integrates chemoreceptor signals. Using multi-electrode arrays and spike train analysis, we measured the activities of 340 raphe neurons together with the phrenic nerve during sequential transient central and/or peripheral chemoreceptor stimulation via CO2-saturated saline injections in vertebral and carotid arteries, respectively, in 11 decerebrate vagotomized cats. A subset of 267 neurons tested with both stimuli responded to either central (n=44) or peripheral (n=42) chemoreceptor challenges, but not both. Of 66 cells that did respond to both, 30 responded with firing rate changes in the same direction (14 INC, 16 DEC). Among 36 neurons with opposite changes in firing rate, 27 decreased during central chemoreceptor stimulation. Functional connections were detected between neurons in 320 of 4,021 pairs. Central cross-correlogram features include 136 peaks indicative of shared inputs of like sign and 31 troughs suggesting inputs with opposite actions. The presence of offset features, including 88 peaks and 33 troughs, suggest pauci-synaptic excitatory and inhibitory interactions, respectively. The results demonstrate functional convergence of central and peripheral chemoreceptor effects on raphe neuronal assemblies and suggest circuit chains and loops appropriate for gain control of both chemoreceptive inputs to the brainstem cardiorespiratory network. Support: NS19814.

Effects on breathing of focal acidosis at multiple medullary raphe sites in awake goats

To gain insight into why there are chemoreceptors at widespread sites in the brain, mircrotubules were chronically implanted at two or three sites in the medullary raphe nuclei of adult goats (n = 7). After >2 wk, microdialysis (MD) probes were inserted into the microtubules to create focal acidosis (FA) in the awake state using mock cerebral spinal fluid (mCSF) equilibrated with 6.4% (pH = 7.3), 50% (pH = 6.5), or 80% CO(2) (pH = 6.3), where MD with 50 and 80% CO(2) reduces tissue pH by 0.1 and 0.18 pH unit, respectively. There were no changes in all measured variables with MD with 6.4% at single or multiple raphe sites (P > 0.05). During FA at single raphe sites, only 80% CO(2) elicited physiological changes as inspiratory flow was 16.9% above (P < 0.05) control. However, FA with 50 and 80% CO(2) at multiple sites increased (P < 0.05) inspiratory flow by 18.4 and 30.1%, respectively, where 80% CO(2) also increased (P < 0.05) tidal volume, heart rate, CO(2) production, and O(2) consumption. FA with 80% CO(2) at multiple raphe sites also led to hyperventilation (-2 mmHg), indicating that FA had effects on breathing independent of an increased metabolic rate. We believe these findings suggest that the large ventilatory response to a global respiratory brain acidosis reflects the cumulative effect of stimulation at widespread chemoreceptor sites rather than a large stimulation at a single site. Additionally, focal acidification of raphe chemoreceptors appears to activate an established thermogenic response needed to offset the increased heat loss associated with the CO(2) hyperpnea.

Effects on breathing in awake and sleeping goats of focal acidosis in the medullary raphe.

Our aim was to determine the effects of focal acidification in the raphe obscurus (RO) and raphe pallidus (RP) on ventilation and other physiological variables in both the awake and sleep states in adult goats. Through chronically implanted microtubules, 1) a focal acidosis was created by microdialysis of mock cerebrospinal fluid (mCSF), equilibrated with various levels of CO2, and 2) medullary extracellular fluid (ECF) pH was measured by using a custom-made pH electrode. Focal acidosis in the RO or RP, by dialyzing either 25 or 80% CO2 (mCSF pH approximately 6.8 or 6.3), increased (P < 0.05) inspiratory flow by 8 and 12%, respectively, while the animals were awake during the day, but not at night while they were awake or in non-rapid eye movement sleep. While the animals were awake during the day, there were also increases in heart rate and blood pressure (P < 0.05) but no significant change in metabolic rate or arterial Pco2. Dialysis with mCSF equilibrated with 25 or 80% CO2 reduced ECF pH by the same amount (25%) or three times more (80%) than when inspired CO2 was increased to 7%. During CO2 inhalation, the reduction in ECF pH was only 50% of the reduction in arterial pH. Finally, dialysis in vivo only decreased ECF pH by 19.1% of the change during dialysis in an in vitro system. We conclude that 1) the physiological responses to focal acidosis in the RO and RP are consistent with the existence of chemoreceptors in these nuclei, and 2) local pH buffering mechanisms act to minimize changes in brain pH during systemic induced acidosis and microdialysis focal acidosis and that these mechanisms could be as or more important to pH regulation than the small changes in inspiratory flow during a focal acidosis.

CO2 dialysis in nucleus tractus solitarius region of rat increases ventilation in sleep and wakefulness.

To evaluate the function of widely distributed central chemoreceptors during sleep and wakefulness in the rat, we focally stimulate single chemoreceptor sites during naturally occurring sleep-wake cycles by microdialysis of artificial cerebrospinal fluid equilibrated with 25% CO2. In retrotrapezoid nucleus, this increased ventilation (tidal volume) by 24% only in wakefulness (Li A, Randall M, and Nattie E. J Appl Physiol 87: 910-919, 1999). In caudal medullary raphé, it increased ventilation (frequency) by 15-20% only in sleep (Nattie EE and Li A. J Appl Physiol 90: 1247-1257, 2001). Here, in nucleus tractus solitarius (NTS), focal acidification significantly increased ventilation by 11% in sleep and 7% in wakefulness rostrally (n = 5) and by 16% in sleep and 28% in wakefulness caudally (n = 5). The sleep-wake cycle was unaltered. Dialysis with 5% CO2 had no effect. Dialysis with 50% CO2 caudally did not further stimulate ventilation but did disrupt sleep. Central chemoreceptors in the NTS affect breathing in both sleep and wakefulness. The threshold for arousal in caudal NTS is greater than that for the stimulation of breathing.

Central chemosensitivity, sleep, and wakefulness

Neurons in many regions of the lower brain are chemosensitive in vitro. Focal acidification of these same and other regions in vivo can stimulate breathing indicating the presence of chemoreception. Why are there so many sites for central chemoreception? This review evaluates data obtained from unanesthetized rats at three central chemoreceptor sites, the retrotrapezoid nucleus (RTN), the medullary raphé, and the nucleus tractus solitarius (NTS) and extends ideas concerning two hypotheses, which were recently formulated (Nattie, E., 2000. Respir. Physiol. 122, 223–235). (1) The high overall sensitivity of the respiratory control system in the unanesthetized state to small increases in arterial CO 2 relies on an additive or greater effect of these multiple chemoreceptor sites. (2) Chemoreceptor sites can vary in effectiveness dependent on the state of arousal. These ideas fit into a more speculative and general hypothesis that central chemoreceptors are organized in a hierarchical manner as proposed for temperature sensing and thermoregulation (Satinoff, E., 1978. Science 201, 16–22). The presence of a number of chemosensitive sites with varying thresholds, sensitivity, and arousal dependence provides finely tuned control and stability for breathing.

CO2 dialysis in the medullary raphe of the rat increases ventilation in sleep

Central chemoreceptors are widespread within the brain stem. We hypothesize that function at different sites varies with arousal state. In unanesthetized rats, we produced focal acidification at single sites by means of microdialysis using artificial cerebrospinal fluid equilibrated with 25% CO2. Tissue acidosis, measured under anesthesia, is equivalent to that observed with 63 Torr end-tidal PCO2 and is limited to 600 microm. Focal acidification of the retrotrapezoid nucleus increased ventilation by 24% only in wakefulness via an increase in tidal volume (Li A, Randall M, and Nattie E. J Appl Physiol 87: 910-919, 1999). In this study of the medullary raphe, the effect of such focal acidification was in sleep (defined by electroencephalographic and electromyographic criteria): ventilation and frequency increased by 15-20% in non-rapid eye movement sleep, and frequency increased by 15% in rapid eye movement sleep. There was no effect in wakefulness. Chemoreception in the medullary raphe appears to be responsive in sleep. Central chemoreceptors at two different locations appear to vary in effectiveness with arousal state.

Central chemosensitivity, sleep, and wakefulness

Central chemoreceptors are widespread within the brainstem. This conclusion is based on in vitro evidence of neurons from many locations that are responsive to pH changes and on in vivo evidence of respiratory responses to focal acidification at these same locations. Why are there so many chemoreceptor sites? As one possibility we hypothesize that function at different sites varies with arousal state. In unanesthetized rats, we produce focal acidification at single sites with microdialysis (probe tip = 1 mm×240 μm diameter) using artificial cerebrospinal fluid equilibrated with 25% CO2. Tissue acidosis, measured at the region of the retrotrapezoid nucleus (RTN) in unanesthetized rats, is approximately equivalent to that observed with end-tidal PCO2 = 7–8 mmHg above the eupneic value. Focal acidification of the retrotrapezoid nucleus (RTN) increased ventilation by 24% only in wakefulness via an increase in tidal volume [1]. In the medullary raphe, the excitatory effect of such focal acidification was observed only in sleep (defined by EEG and EMG criteria); ventilation and frequency increased by 15–20% in NREM sleep and frequency by 15% in REM sleep. There was no effect in wakefulness [2]. In our most recent study, focal acidification of nucleus tractus solitarius (NTS) increased ventilation by up to 20% in both wakefulness and NREM sleep due to an effect on both tidal volume and frequency. The increase in ventilation is greater with focal acidification in the caudal part of NTS at the level of the area postrema. Acidification of more rostral parts of the NTS produced variable results. The medullary raphe contributes to chemoreception in sleep, the RTN in wakefulness, and the cNTS in both sleep and wakefulness. Central chemoreceptors at these three different locations do appear to vary in effectiveness with arousal state. The response at each site is only a fraction of the response attributable to all sites being stimulated at the same intensity. Hypocapnia resulting from single site stimulation can lower the contribution of other sites minimizing the apparent effectiveness of single site stimulation.

Chemoreception and tonic drive in the retrotrapezoid nucleus (RTN) region of the awake rat: bicuculline and muscimol dialysis in the RTN.

In anesthetized animals, focal acidification of a portion of the rostral ventrolateral medulla (RVLM), the retrotrapezoid nucleus (RTN), increases respiratory output3, 6. We define the RTN region to include the RTN proper, described by retrograde tracing experiments as lying ventral to the facial nucleus19and adjacent neurons of the parapyramidal region and the juxta-facial portion of the nucleus paragigantocellularis lateralis. Focal disruption of the RTN region by cooling2 or by neurotoxin injection10,12,13 substantially reduces baseline respiratory output and the response to systemic hypercapnia. The RTN region can provide a tonic drive to breathe and is a site of central chemoreception.KeywordsRostral Ventrolateral MedullaDialysis ProbeCentral ChemoreceptorDartmouth Medical SchoolVentral Respiratory GroupThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

CO(2) microdialysis in retrotrapezoid nucleus of the rat increases breathing in wakefulness but not in sleep.

Central chemoreceptors are widespread within the brain stem. We suggest that their function at some sites may vary with the state of arousal. In this study, we tested the hypothesis that the function of chemoreceptors in the retrotrapezoid nucleus (RTN) varies with sleep and wakefulness. In unanesthetized rats, we produced focal acidification of the RTN by means of a microdialysis probe (tip containing the semipermeable membrane = 1-mm length, 240-microm diameter, and 45-nl volume). With the use of a dialysate equilibrated with 25% CO(2), the tissue pH change (measured in anesthetized animals) was 1) limited to within 550 microm of the probe and, 2) at the probe tip, was equivalent to that observed with end-tidal PCO(2) of 63 Torr. This focal acidification of the RTN increased ventilation significantly by 24% above baseline, on average, in 13 trials in seven rats only during wakefulness. The effect was entirely due to an increase in tidal volume. During sleep defined by behavioral criteria, ventilation was unaffected, on average, in 10 trials in seven rats. During sleep, the chemoreceptors in the RTN appear to be inactive, or, if active, the respiratory control system either is not responding or is responding with very low gain. Because ventilation is increased during sleep with all central chemoreceptor sites stimulated via systemic CO(2) application, other central chemoreceptor locations must have enhanced effectiveness.