Ventilatory response to hypercapnia and hypoxia after extensive lesion of medullary serotonergic neurons in newborn conscious piglets.

Acute inhibition of serotonergic (5-HT) neurons in the medullary raphé (MR) using a 5-HT(1A) receptor agonist had an age-dependent impact on the "CO(2) response" of piglets (33). Our present study explored the effect of chronic 5-HT neuron lesions in the MR and extra-raphé on the ventilatory response to hypercapnia and hypoxia in piglets, with possible implications on the role of 5-HT in the sudden infant death syndrome. We established four experimental groups. Group 1 (n = 11) did not undergo any treatment. Groups 2, 3, and 4 were injected with either vehicle or the neurotoxin 5,7-dihydroxytryptamine in the cisterna magna during the first week of life (group 2, n = 9; group 4, n = 11) or second week of life (group 3, n = 10). Ventilation was recorded in response to 5% CO(2) (all groups) and 12% O(2) (group 2) during wakefulness and sleep up to postnatal day 25. Surprisingly, the piglets did not reveal changes in their CO(2) sensitivity during early postnatal development. Overall, considerable lesions of 5-HT neurons (up to 65% decrease) in the MR and extra-raphé had no impact on the CO(2) response, regardless of injection time. Postlesion raphé plasticity could explain why we observed no effect. 5,7-Dihydroxytryptamine-treated males, however, did present a lower CO(2) response during sleep. Hypoxia significantly altered the frequency during sleep in lesioned piglets. Further studies are necessary to elucidate the role of plasticity, sex, and 5-HT abnormalities in sudden infant death syndrome.

Spontaneous arousals during quiet sleep in piglets: a visual and wavelet-based analysis.

STUDY OBJECTIVES: The objectives of the study were to characterize spontaneous arousals during NREM sleep in piglets and to compare two methods of identifying these events: a "visual" technique using spectral analysis and an automated technique using wavelets. Our goal was to understand the benefits and limits of these methods when applied to sleep in human infants. DESIGN: Arousals were identified by evaluating rapid changes in EEG low frequency activity, blood pressure (BP), and heart rate (HR). A cortical arousal was defined as a rapid decrease in EEG low frequency activity. An autonomic arousal was defined by a transient increase in heart rate or a transient change in mean arterial BP (MAP). SETTING: Laboratory study in sleeping and awake piglets. PARTICIPANTS: Five 1-2 week old piglets. INTERVENTIONS: Chronically instrumented with a femoral arterial line, EEG, EOG, EMG electrodes, and a micro-dialysis probe with its tip located in the rostral ventral medulla. Artificial CSF (aCSF) was dialyzed into the RVM throughout the experiments Measurements: For the visual analysis, the average delta power (0.5-4 Hz) for each 5-second epoch was determined using spectral analysis. MAP and HR were analyzed in 1-second bins. Video images were analyzed for body movements and eye openings. Transient changes in blood pressure, HR, and delta power were then visually identified. For the wavelet analysis, a quantitative, automated technique with a defined "wakefulness threshold" was used to identify rapid decreases in EEG low frequency activity and the rate of change of MAP. RESULTS: Using the visual method, 117 episodes associated with stereotypical hemodynamic, EEG, and behavioral changes (startle) were identified. Seventy five events occurred in isolation or were first in a series of "multiple" events, 41 "multiple" events were defined as events occurring <20 seconds following a previous event. Eighteen events were associated with the termination of apnea. In isolated events or those occurring first in a series, the onset of changes in HR and BP clearly preceded the decrease in EEG amplitude and delta power. Using wavelet analysis, 73 EEG arousals and 115 MAP transients were identified independently; 62% of the EEG events were associated with a transient change in MAP and HR, and in these cases the onset of the hemodynamic events preceded EEG arousals. EEG arousals and MAP transients, however, also occurred alone and not associated with a stereotypical pattern of a startle, changes in MAP and HR and the EEG. CONCLUSIONS: Many of these spontaneous arousals represent integrated EEG, hemodynamic, and behavioral processes similar to arousal phenomena described in adult rats and human infants, but the pattern of spontaneous arousals appears to be more heterogeneous than has been described for arousals induced by exogenous stimuli. Both the visual and wavelet analysis identified these events, but the wavelet technique has the potential advantage of better time resolution and automation of the analysis.

The effects of a GABA(A) agonist in the rostral ventral medulla on sleep and breathing in newborn piglets.

STUDY OBJECTIVES: Abnormalities in the rostral ventral medulla (RVM) in human infants may contribute to the etiology of the sudden infant death syndrome (SIDS) or a subset of SIDS, by interfering with cardiorespiratory and arousal responses to physiological stimuli often encountered during sleep. The purpose of this study was to determine whether inhibition of groups of neurons in the RVM in newborn piglets would alter sleep and/or the sleep-modulation of breathing. We hypothesized that inhibition of neurons in the RVM would produce less wakefulness or increase the low frequency power (delta) during Quiet sleep. DESIGN: Unanesthetized piglets were studied in a whole-body plethysmograph. Artificial cerebral spinal fluid (aCSF) or the GABAA agonist, muscimol, was dialyzed into the RVM for 40 minutes after a control period consisting of aCSF dialysis. Sleep was analyzed using a combination of EEG spectral analysis and behavioral observations. SETTING: N/A. PARTICIPANTS: N/A. INTERVENTIONS: N/A. MEASUREMENTS AND RESULTS: Cardiorespiratory variables varied with state. Dialysis of neither aCSF nor muscimol into the RVM resulted in alterations in resting respiration, BP, HR, or VO2 or their modulation by state. Compared to control dialysis with aCSF, muscimol dialysis caused dramatic effects on sleep architecture. Sleep cycling was abolished in some experiments, whereas in others there were decreases in low-frequency EEG activity or delta power. The animals in which sleep cycling ceased continued in a perpetual state of drowsiness interspersed with periods of wakefulness. CONCLUSIONS: We conclude that dialysis of muscimol into the RVM has little effect on resting breathing, blood pressure, or heart rate or their modulation by state, but interferes with normal sleep architecture. We speculate that abnormalities in the ventral medulla may alter sleep cycling or interfere with arousal mechanisms, thus contributing to the etiology of at least a subset of SIDS.

Diethyl pyrocarbonate (an imidazole binding substance) inhibits rostral VLM CO2 sensitivity.

Diethyl pyrocarbonate (DEPC) has been useful in vitro as an agent relatively specific for binding to imidazole of histidine. Administered via the cisterna magna DEPC inhibits central chemosensitivity in conscious rabbits, supporting the alphastat hypothesis for central chemoreceptor function. In this study I have applied DEPC via 1 X 3 mm cottonoid pledgets to each of the three ventrolateral medulla (VLM) chemosensitive areas in glomectomized, vagotomized, paralyzed, and servo-ventilated alpha-chloralose-urethan-anesthetized cats. CO2 responses measured by integrated phrenic nerve output were evaluated before and after DEPC application. A dose of 40 mmol/l applied to the rostral chemosensitive area increased the CO2 threshold (5.3%) and significantly decreased (P less than 0.03; Wilcoxon sign rank test) the initial slope (-43%) and the maximum (-41%) of the CO2 response. No significant effects were observed with DEPC application in the intermediate or caudal areas. Treatment with 40 mmol/l hydroxylamine immediately after DEPC in the rostral area prevented the effects supporting the interpretation that imidazole was the reactant with DEPC. The results are consistent with the hypothesis that imidazole-histidine is involved in the mechanism of central chemoreception and indicate that only the rostral area utilizes a DEPC inhibitable mechanism.

The alphastat hypothesis in respiratory control and acid-base balance.

This selective review 1) evaluates recent interpretations that broaden the definition of the alphastat hypothesis, 2) proposes that central chemoreception and acid-base regulation via ion transport involve proteins conforming to the alphastat hypothesis, and 3) describes, using recent evidence, possible candidates for these proteins. The alphastat hypothesis states that proteins that contain appropriate function-determining titratable groups maintain a constant charge state and unaltered function with temperature-dependent pH changes but can be very sensitive to isothermal pH changes. Appropriate groups, e.g., imidazole histidine, are determined by the pK and the effect of temperature on the pK. The hypothesis explains how protein structure and function can be conserved among a diversity of vertebrate and invertebrate pH values. It also suggests a mechanism for sensing or regulating temperature-independent pH changes, e.g., in central chemosensitivity and transmembrane ion exchange. Possible candidates for such alphastat-conforming proteins include two, the glutamate receptor and the Na(+)-H+ antiporter, for which recent evidence indicates the presence of numerous histidines at probable function-determining sites and demonstrates pH sensitivity inhibitable by the histidine blocker diethylpyrocarbonate (DEPC).

Diethyl pyrocarbonate inhibits rostral ventrolateral medullary H+ sensitivity.

Diethyl pyrocarbonate (DEPC), an acylating agent that reacts with imidazole-histidine in vitro, inhibits CO2 sensitivity when applied by pledget to the rostral chemosensitive area on the ventrolateral medullary (VLM) surface in glomectomized, chloralose-urethan-anesthetized cats. In this study similar application of DEPC inhibits the phrenic nerve response to CO2 expressed as a function of VLM [H+] measured by surface pH electrode. Attempts to evaluate direct chemoreceptor stimulation by HCL-soaked surface pledgets proved difficult, but rostral DEPC did inhibit the response to intravenous infusion of HCl. As previously reported, the CO2 and intravenous H+ responses are not a unique function of the VLM [H+]. DEPC had similar inhibitory effects on both the CO2 and the intravenous H+ responses, suggesting that the difference between them may reflect more the orientation or accessibility of the central chemoreceptor than a different mechanism for sensing CO2 vs. H+. DEPC did not alter the phrenic nerve response to hypoxia, indicating that DEPC effects on central chemoreception are not the result of a generalized inhibitory process. The results support the hypothesis that imidazolehistidine is involved at the rostral area with chemoreception of both CO2 and H+.

Central vs. peripheral chemoreceptors in ventilatory stimulation by Hacetate.

Intravenous infusion of Hacetate in conscious rabbits induces a greater decrease in cerebrospinal fluid (CSF) [HCO3-] and arterial CO2 partial pressure (PaCO2) than does HCl, HNO3, or Hacetate. To test whether acetate per se can stimulate central chemoreceptors, HCl- or Hacetate-acidified mock CSF was infused via the cisterna magna in conscious rabbits with catheters preimplanted under anesthesia. HCl infusion induced a greater decrease in PaCO2 refuting this hypothesis. To evaluate the role of the carotid body HCl and Hacetate were infused intravenously in an intact (CB+) and a chemodenervated group (CB-). In CB+ rabbits Hacetate infusion produced a greater decrease in PaCO2. In CB- rabbits, the fractional decrease in arterial PaCO2 was less for both acids compared with that of the CB+ rabbits, but it was significantly greater for Hacetate infusion (21.2 +/- 2.5%, mean +/- SE) than for HCl infusion (14.5 +/- 1.8%). Thus the carotid body is not necessary for the greater Hacetate ventilatory stimulation. The working hypothesis is that nonionic diffusion of Hacetate into brain or acetate replacement of HCO3- in CSF production lowers [HCO3-] near central chemoreceptors.

CSF acid-base regulation and ventilation in iso- and hypocapnic Hacetate acidosis.

Intravenous infusion in conscious rabbits of Hacetate decreases both arterial CO2 partial pressure PaCO2 and cerebrospinal fluid (CSF) HCO3- more than observed with HCl or HNO3 infusion. These acids did not affect CSF HCO3- in isocapnic conditions, and this study asks whether Hacetate infusion will do so. Arterial, central venous, and cisterna magna catheters were implanted in pentobarbital-anesthetized rabbits and all subsequent measurements were performed in the conscious state. Hacetate was infused intravenously over 6 h to decrease plasma HCO3- the same amount in a group allowed to decrease its PaCO2 in response to the acid (hypocapnic) and one in which PaCO2 was maintained at control levels (isocapnic). CSF HCO3- decreased significantly in isocapnia, although the change was less than in hypocapnia. Stoichiometrically by 6 h the measured CSF HCO3- change was balanced by an increase in acetate in hypocapnia and the sum of an increase in acetate and a decrease in chloride in isocapnia. Mechanistically, net acetate entry into CSF appears to involve an exchange for chloride as proposed for NO3-/Cl- and a process that lowers CSF HCO3-. This process could be competitive replacement of HCO3- by acetate in the CSF production mechanism or nonionic diffusive entry of Hacetate into CSF with subsequent titration of HCO3-. The decreases in CSF HCO3- result from the acetate mechanism and the hypocapnic effect on Cl- and HCO3-. The greater ventilatory response results from the greater CSF acidification or a specific effect of acetate per se.

Fluorescence location of RVLM kainate microinjections that alter the control of breathing.

Kainic acid (4.7 mM) applied to the rostral ventrolateral medulla (RVLM) surface decreases phrenic output, CO2 sensitivity, and blood pressure in chloralose-urethan-anesthetized, vagotomized, paralyzed, glomectomized, servoventilated cats. In this study using the same preparation, bilateral 50- to 100-nl kainate injections just below the RVLM surface better localized these responses topographically. The physiological responses to unilateral 10-nl kainate injections were then correlated with anatomic location determined by fluorescent microbeads (0.5 micron diam). Many sites were associated with no effect, a few rostral and caudal sites with increased phrenic activity, and cluster of sites with decreased phrenic activity often to apnea, decreased CO2 sensitivity, and decreased responses to carotid sinus nerve stimulation. Blood pressure was unaffected. These sites, within 400 microns of the surface, were ventral to the facial nucleus, ventrolateral to the nucleus paragigantocellularis lateralis, caudal to the superior olive, and rostral to the retrofacial nucleus. They appeared to be within the recently described retrotrapezoid nucleus, which contains cells with respiratory-related activity and projections to the dorsal and ventral respiratory groups. Cells within this site appear able to provide tonic input to respiration and to affect peripheral and central chemoreception.

Ventral medulla sites of muscarinic receptor subtypes involved in cardiorespiratory control.

Bilateral 10-nl injections of the muscarinic (M1) receptor antagonist pirenzepine (PZ) and the M3-antagonist 4-diphenylacetoxy-N-methylpiperidine (DAMP) just below the surface of the rostral ventrolateral medulla (VLM) in anesthetized, paralyzed, vagotomized, glomectomized, servo-ventilated cats decreased base-line phrenic activity and CO2 sensitivity but not blood pressure. Unilateral 10-nl injections of PZ, DAMP, or the M2-antagonist AFDX-116 resulted in the following. AFDX-116 greater than PZ greater than DAMP decreased blood pressure. DAMP greater than PZ greater than AFDX-116 decreased base-line phrenic activity and the CO2 response slope. Many injections had no effect, and some affected only one variable. The injection sites were within 700 microns of the VLM surface extending from the caudal aspect of the superior olive through a region ventromedial to the facial nucleus to the rostral aspect of the inferior olive. In a small number of experiments the response to carotid sinus nerve stimulation was affected by some injections of each antagonist. In the rostral VLM cardiovascular regulation involves M2-receptors, whereas respiratory regulation involves predominantly the M3-subtype. Neurons with these receptors appear to be closely intermingled.

Effects of amiloride and diethyl pyrocarbonate on CSF HCO-3 and ventilation in hypercapnia.

Amiloride (10(-3) M), a Na+-H+ countertransport inhibitor, infused into the cisterna magna (10 microliter/min for 40 min) of ketamine-xylazine-anesthetized rabbits decreased the cerebrospinal fluid (CSF) HCO3- response to 3 h of hypercapnia [arterial PCO2 (PaCO2) = 60 Torr] by 21.6% (mean delta CSF [HCO3-]/delta PaCO2 0.232 vs. 0.296 mmol.l-1.Torr-1, P less than 0.05). Diethyl pyrocarbonate (DEPC, 10(-3) M), a histidine-blocking agent, infused into the cisterna magna decreased the CSF HCO3- response to hypercapnia by 25.3% (mean delta CSF [HCO3-]/delta PaCO2, 0.230 vs. 0.308 mmol.l-1.Torr-1, P less than 0.02). DEPC is known to inhibit the ventilatory response to hypercapnia (E. Nattie. Respir. Physiol. 64: 161-176, 1986) by a direct effect at the ventrolateral medulla (E. Nattie. J. Appl. Physiol. 61: 843-850, 1986). In this study amiloride had no significant effect on the ventilatory response to hypercapnia. The interpretation is that a Na+-H+ countertransport protein, perhaps with a histidine at a key location, is involved in CSF acid-base regulation and that amiloride appears to have no effects on the chemoreception process. DEPC appears to have effects on chemoreception and on CSF acid-base regulation.

Central chemoreception in the region of the ventral respiratory group in the rat.

We injected acetazolamide (AZ; 5 x 10(-6) M, 1 nl) into the region of the ventral respiratory group (VRG) of anesthetized paralyzed ventilated rats. Control injections (mock cerebrospinal fluid, n = 6, or the inactive AZ analogue 2-acetylamino-1,3, 4-thiadiazole-5-sulfon-t-butylamide, n = 6) did not increase the integrated phrenic neurogram [phrenic nerve amplitude (PNA)]. The AZ injections produced a focal region of tissue acidosis with a radius < 300-400 microns and are used as a probe for sites of central chemosensitivity. Injection location is determined by anatomic analysis. Of 22 VRG injections of AZ, 14 increased the amplitude of the PNA over 15-90 min; 8 had no effect. In 17 cases, we measured medullary tissue pH at the injection center and/or at a distant site and reaffirmed the size of the acidotic region produced by such small AZ injections. Of injections with pH electrodes within 300-400 microns of the injection center, all responders showed an acid pH; three nonresponders showed an acid pH, and one an alkaline pH. In a subgroup of five rats, at VRG sites with known respiratory effects identified by prior glutamate injection (10 nl, 100 mM), all subsequent AZ injections produced a PNA response. Simultaneous measurement of PNA and tissue pH responses at the injection center of eight rats did not show a uniform correlation in time; initially, both changed with a similar time course, but PNA recovered more quickly. We conclude that 1) the region of the VRG contains sites of ventilatory chemoreception, 2) ineffective AZ injections do produce a tissue acidosis but at sites with minimal impact on breathing, and 3) tissue pH does not uniquely represent the chemoreceptor stimulus.

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.

Retrotrapezoid nucleus glutamate injections: long-term stimulation of phrenic activity.

In chloralose-urethan anesthetized, paralyzed, vagotomized, glomectomized, and servo-ventilated cats we examined the effects of 10 nl of glutamate (10 mM, 100 mM, and 1 M) injected unilaterally over 60 s into the region of the retrotrapezoid nucleus (RTN). Seven 10 mM glutamate injections produced no consistent effects on the amplitude of the integrated phrenic nerve signal, respiratory cycle duration, or blood pressure. Ten 100 mM injections consistently increased integrated phrenic amplitude significantly from a baseline average of 31 +/- 2% (SE) of maximum to a peak response average of 50 +/- 3% of maximum. This effect was long lasting (45.6 +/- 8.6 min). Blood pressure responses were variable. Seven 1 M glutamate injections consistently decreased integrated phrenic amplitude significantly from a baseline average for all injections of 29 +/- 3% of maximum to a peak average of 20 +/- 5% of maximum. Respiratory cycle duration and blood pressure responses were variable. Prior injection into the RTN of 10 nl of 100 mM kynurenic acid attenuated the subsequent response of the integrated phrenic amplitude to injection of 10 nl of glutamate at the same site. Comparison of glutamate (10 nl, 100 mM) injected over 60 s vs. 30 ms showed that the prolonged increase in phrenic activity was related to the longer-duration (60-s) injections and that RTN single units were stimulated for up to 5 min on average after the 60-s injection with one unit showing an increase in firing rate over 40 min. After the 30-ms injection, integrated phrenic amplitude and RTN unit mean firing rate were increased for the first two breaths and at 5 min after the injection. We conclude that glutamate injected into the RTN increases local single-unit firing rate and the amplitude of the integrated phrenic activity. Injections made over 60 s result in prolonged phrenic stimulation and, in some cases, in RTN single-unit firing rate.

DIDS decreases CSF HCO3- and increases breathing in response to CO2 in awake rabbits.

An inhibitor of the HCO3-/Cl- exchange carrier protein, 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) or vehicle was infused in mock cerebrospinal fluid (CSF) via the cisterna magna in conscious rabbits at 10 mumol/l for 40 min at 10 microliter/min. Neither treatment had any effect over 2-5 h on the non-CO2-stimulated CSF ion values or blood gases. With CO2 stimulation such that arterial PCO2 (PaCO2) was increased 25 Torr over 3 h, DIDS treatment significantly decreased the stoichiometrically opposite changes in CSF [HCO3-] and [Cl-] that normally accompany hypercapnia and reflect ionic mechanisms of CSF pH regulation. Expressed as delta CSF [HCO3-]/delta PaCO2, DIDS treatment decreased the CSF ionic response by 35%. In a separate paired study design DIDS administration via the same protocol had no effect on resting ventilation but significantly increased the ventilation and tidal volume responses to a 28-Torr increase in PaCO2. Expressed as change in minute ventilation divided by delta PaCO2, DIDS treatment produced a 39.6% increase. The results support the concept of a DIDS-inhibitable anion exchange carrier being involved in CSF pH regulation in hypercapnia and suggest a DIDS-related effect on the ventilatory response to CO2.

Rat retrotrapezoid nucleus iono- and metabotropic glutamate receptors and the control of breathing.

We injected 10 nl (unilateral) of glutamate receptor antagonists or agonists into the region of the retrotrapezoid nucleus and measured the phrenic nerve and blood pressure responses. The rats were chloralose-urethan anesthetized, paralyzed, vagotomized, and ventilated, and each injection location was verified anatomically. Integrated phrenic amplitude was most reliably affected. The N-methyl-D-aspartic acid (NMDA) antagonists 2-amino-5-phosphonopentanoic acid and 6-cyano-7-nitroquinoxaline-2,3-dione (which affects both NMDA and non-NMDA receptors) both decreased baseline eucapnic phrenic amplitude and the CO2 response. Glutamate increased phrenic amplitude in a dose-dependent manner, an effect blocked by prior injection of the NMDA and non-NMDA antagonists at the same site. The response duration depended on the duration of the glutamate injection: responses to 3-s injections lasted a few minutes, and responses to 60-s injections lasted for > 30 min. The long-lasting effect was reproduced by injection of the metabotropic agonist 1(S),3(R)-aminocyclopentanedicarboxylic acid at 0.01-0.02 times the glutamate dose. We conclude that the rat retrotrapezoid nucleus has an endogenous source of glutamate that maintains eucapnic phrenic output and allows expression of the CO2 response. NMDA and possibly non-NMDA receptors are involved. Their stimulation increases phrenic output via ionotropic and metabotropic receptor processes with the latter resulting in long-lasting phrenic stimulation.

Focal central chemoreceptor sensitivity in the RTN studied with a CO2 diffusion pipette in vivo.

We describe and use a CO2 diffusion pipette to produce a quickly reversible focal acidosis in the retrotrapezoid nucleus region of the rat brain stem. No tissue injection is made. Instead, artificial cerebrospinal fluid (aCSF) equilibrated with CO2 circulates within the micropipette, providing a source for continued CO2 diffusion into the tissue from the pipette tip. Tissue pH electrodes show the acidosis is limited to 500 micron from the tip. In controls (aCSF equilibrated with air), 1-min pipette perfusions increased tissue pH slightly and decreased phrenic nerve amplitude. In moderate- and high-CO2 groups (aCSF equilibrated with 50 or 100% CO2), 1-min perfusions significantly decreased tissue pH and increased phrenic nerve amplitude in a dose-dependent manner. The responses developed and reversed within minutes. Compared with our prior use of medullary acetazolamide injections to produce a focal acidosis, in this approach the acidosis 1) arises and reverses quickly and 2) its intensity can be varied. This allows study of sensitivity and mechanism. We conclude from this initial experiment that retrotrapezoid nucleus region chemoreceptors operate within the normal physiological range of CO2-induced tissue pH changes.

Prolonged stimulation of respiration by brain stem metabotropic glutamate receptors.

Stimulation of metabotropic glutamate receptors (mGluRs) in the retrotrapezoid nucleus (RTN) of chloralose-urethan-anesthetized rats by the mGluR agonist (1S,3R)-aminocyclopentanedicarboxylic acid [(1S,3R)-ACPD; 10 nl, 1 mM] increases integrated phrenic nerve amplitude (PNA) for > 60 min. Here we ask if the mGluR antagonist (+-)-alpha-methyl-4-carboxyphenylglycine [(+-)-MCPG] can block this effect. Using multibarreled micropipettes, we first identified RTN sites that affect respiration by noting short-lived stimulation of PNA produced by brief-duration injection of glutamate (10 nl, 100 mM), a presumed ionotropic receptor response. We then injected the active or inactive isomer of (+-)-MCPG (10 nl, 10 mM) followed by (1S,3R)-ACPD or by a long-duration (60-s) injection of glutamate (10 nl, 100 mM) at the same site. Each injection location was verified anatomically. The active antagonist (+-)-MCPG itself had no significant effect on PNA, but it blocked 1) subsequent (1S,3R)-ACPD-induced PNA stimulation for 99 +/- 11 (SE) min and 2) the PNA response to 60-s glutamate injection for 66 +/- 6 min. The inactive form (-)-MCPG had no effect on (1S,3R)-ACPD-induced PNA stimulation. We conclude that 1) RTN mGluRs may be involved in respiratory control, 2) RTN mGluRs are not active in eupnea, and 3) stimulation of RTN mGluRs may require prolonged glutamate release.

Retrofacial lesions: effects on CO2-sensitive phrenic and sympathetic nerve activity.

We made unilateral chemical (10- or 50-nl microinjections; 4.7 mM kainic acid) or electrolytic (5-15 mA; 15 s) lesions in a region of the rostral ventrolateral medulla (VLM) caudal to the retrotrapezoid nucleus in 10 decerebrate, paralyzed, vagotomized, and servo-ventilated cats. The lesions were 3.0-4.2 mm lateral to the midline, within 2 mm caudal to the facial nucleus, and within 2.5 mm of the VLM surface. Four control injections (mock cerebrospinal fluid and fluorescent beads alone) produced small and inconsistent effects over 3-5 h. The predominant effect of the lesions was a significant decrease in baseline integrated phrenic nerve amplitude (PNA) (apnea in 2 cases), total respiratory cycle duration, and the response to increased CO2 (slope < 15% of control in 3 cases). The respiratory-related peak amplitude of the integrated sympathetic signal, blood pressure, and the sympathetic nerve activity response to CO2 were also decreased after the majority of lesions. Not all lesions produced all effects, and some lesions resulted in increased PNA and respiratory cycle duration. The lesioned region appears functionally to represent a caudal extension of the retrotrapezoid nucleus containing neurons necessary for normal baseline PNA and CO2 sensitivity. In addition, it contains neurons involved in the determination of resting respiratory frequency and normal sympathetic activity and blood pressure. The pattern of mixed responses among animals suggests that a heterogeneity of function is present within a relatively small VLM region.

RTN TRH causes prolonged respiratory stimulation.

We injected thyrotropin-releasing hormone (TRH; 10 nl; 0.25, 0.5, 1.0, or 10 mM), its inactive free acid form (TRHOH; 1 mM), or a metabolite with low TRH-receptor binding affinity, histidine-proline diketopiperazine (cHP; 1 mM), into the retrotrapezoid nucleus of anesthetized rats. Injection location was verified by anatomic analysis. Lower doses (0.25-0.5 mM) significantly increased both the product of integrated phrenic amplitude and frequency (Phr . f) and f for 20-30 min compared with artificial cerebrospinal fluid control injections. Higher doses (1. 0-10 mM) produced greater and long-lasting stimulation of Phr . f, Phr, and f and of blood pressure. This stimulation reached values 150% of baseline and durations of 270 min after a single injection. TRHOH (1 mM ) or cHP (1 mM) had no effect on Phr but increased f, as did 1 mM TRH. We conclude that TRH has a very powerful stimulatory effect in the retrotrapezoid nucleus region on Phr . f, with the Phr response seemingly specific for TRH receptors. Similar responses of f to TRHOH and cHP suggest it may be nonspecific.