Edward J. Zuperku

Modulation of the synaptic drive to respiratory premotor and motor neurons.

The characteristics of GABAergic inhibitory modulation of respiratory bulbospinal neuronal activity and short-term potentiation (STP) of phrenic motoneuronal activity were studied. Extracellular unit recording and picoejection techniques in anesthetized dogs showed that both the spontaneous rhythmic and reflexly induced discharge patterns of inspiratory (I) and expiratory (E) premotor neurons were proportionately amplified by the localized application of picomole amounts of bicuculline (Bic), a competitive GABAA antagonist. Intracellular recording and paired-pulse stimulation techniques in anesthetized rats demonstrated an STP of phrenic motor output that appears to be mediated by NMDA receptors and is associated with facilitation of EPSPs and prolonged depolarization of individual phrenic motoneurons. We speculate that both GABAergic gain modulation of premotor neuronal activity and NMDA-mediated STP of phrenic activity may be neural substrates which are involved with the optimization of respiratory and non-respiratory behaviors, via adaptive and/or differential control of breathing.

Effects of Sevoflurane on excitatory neurotransmission to medullary expiratory neurons and on phrenic nerve activity in a decerebrate dog model

Background Sevoflurane is a new volatile anesthetic with a pronounced respiratory depressant effect. Synaptic neurotransmission in canine expiratory bulbospinal neurons is mainly mediated by excitatory N-methyl-d-aspartatic acid (NMDA) receptor input and modulated by inhibitory &ggr;-aminobutyric acid type A (GABAA) receptors. The authors investigated the effect of sevoflurane on these mechanisms in decerebrate dogs. Methods Studies were performed in decerebrate, vagotomized, paralyzed and mechanically ventilated dogs during hypercapnic hyperoxia. The effect of 1 minimum alveolar concentration (MAC; 2.4%) sevoflurane on extracellularly recorded neuronal activity was measured during localized picoejection of the glutamate agonist NMDA and the GABAA receptor blocker bicuculline in a two-part protocol. First, complete blockade of the GABAAergic mechanism by bicuculline allowed differentiation between the effects of sevoflurane on overall GABAAergic inhibition and on overall glutamatergic excitation. In a second step, the neuronal response to exogenous NMDA was used to estimate sevoflurane’s effect on postsynaptic glutamatergic neurotransmission. Results One minimum alveolar concentration sevoflurane depressed the spontaneous activity of 16 expiratory neurons by 36.7 ± 22.4% (mean ± SD). Overall glutamatergic excitation was depressed 19.5 ± 16.2%, and GABAAergic inhibition was enhanced 18.7 ± 20.6%. However, the postsynaptic response to exogenous NMDA was not significantly altered. In addition, 1 MAC sevoflurane depressed peak phrenic nerve activity by 61.8 ± 17.7%. Conclusions In the authors’in vivo expiratory neuronal model, the depressive effect of sevoflurane on synaptic neurotransmission was caused by a reduction of presynaptic glutamatergic excitation and an enhancement of GABAAergic inhibition. The effects on expiratory neuronal activity were similar to halothane, but sevoflurane caused a stronger depression of phrenic nerve activity than halothane.

Pattern formation and rhythm generation in the ventral respiratory group.

1. There is increasing evidence that the kernel of the rhythm‐generating circuitry for breathing is located within a discrete subregion of a column of respiratory neurons within the ventrolateral medulla referred to as the ventral respiratory group (VRG). It is less clear how this rhythm is transformed into the precise patterns appearing on the varied motor outflows.

Gain modulation of respiratory neurons

A possible mechanism underlying adaptive control of the respiratory system is gain modulation of the discharge frequency (F(n)) patterns of medullary respiratory neurons mediated by GABA(A) receptors. Antagonism of GABA(A) receptors with bicuculline results in an F(n) pattern that is an amplified replica of the underlying control pattern. The contours of F(n) patterns remain proportional to one another. Studies suggest that a tonic GABA(A)ergic input constrains the control- and reflexly-induced activities of these neurons to about 35-50% of the discharge rate without this inhibitory input. The pharmacology of this mechanism is unusual in that picrotoxin, a noncompetitive GABA(A) receptor antagonist, does not produce gain modulation, but is able to block the silent phase inhibition (e.g. E phase of an I neuron). Alterations in the amplitude of spike afterhyperpolarizations mediated by Ca(2+) activated K(+) channels also produces gain modulation. This mechanism modulates exogenously- and endogenously-induced neuronal activities, whereas the bicuculline-sensitive GABAergic mechanism modulates only the respiratory-related activities. Thus, these two forms of gain modulation, acting in cascade manner, may provide robust mechanisms for the optimal control of respiratory, as well as other behavioral functions (e.g. coughing, sneezing, vomiting) mediated by respiratory premotor neurons.

Unraveling the mechanism for respiratory rhythm generation

Breathing is generated by a neuronal network located within the caudal brainstem. One area of particular significance for respiratory rhythm generation is the pre-Bötzinger (preBotC) complex in the ventrolateral medulla. An important step towards understanding the cellular and network basis by which neurons within this region generate the respiratory rhythm was made in a recent study by Koshiya and Smith.(1) Using simultaneous image analysis and electrophysiological techniques these authors identified a discrete population of synaptically-coupled pacemaker neurons within the preBotC. They postulated that these neurons constitute the minimal essential network component (kernel) for generating the respiratory rhythm. BioEssays 22:6-9, 2000.

NMDA receptor-mediated transmission of carotid body chemoreceptor input to expiratory bulbospinal neurones in dogs.

1. This study tested the hypothesis that excitatory amino acid receptors mediate the excitatory response of expiratory bulbospinal neurones to carotid body chemoreceptor inputs. 2. Studies were carried out in thiopental sodium anaesthetized, paralysed, ventilated, vagotomized dogs. 3. Brisk, short‐duration chemoreceptor activation was produced by bilateral bolus injections of CO2‐saturated saline (PCO2 > 700 mmHg) into the autoperfused carotid arteries. A pressurized‐reservoir‐solenoid valve system was used to deliver the CO2 bolus injections just prior to the onset of the neural expiratory phase, as determined from the phrenic neurogram, about once per minute. 4. Multibarrelled micropipettes were used to record neuronal unit activity and deliver neurotransmitter agents. Net responses of expiratory bulbospinal neurones to peripheral chemoreceptor activation were determined by subtracting the mean discharge frequencies (Fn) during three control expiratory cycles from the Fn during administration of a CO2 test bolus. The role of excitatory amino acid receptors in mediating this response was determined by comparing the baseline and bolus expiratory neuronal Fn before, during and after the pressure microejection of the NMDA receptor antagonist 2‐amino‐5‐phosphonovalerate (AP5) or the non‐NMDA receptor antagonist 2,3‐dihydroxy‐6‐nitro‐7‐sulphamoyl‐ benzo(f)quinoxaline (NBQX). Ejection rates of AP5 and NBQX were measured by monitoring the movement of the pipette meniscus. 5. AP5 reduced Fn during both the control and bolus cycles, as well as reducing the change in Fn between control and bolus cycles. NBQX had no effect on either baseline or bolus responses. 6. AP5 did not prevent excitation of expiratory bulbospinal neurones by AMPA. Coadministration of AMPA with AP5 prevented the AP5‐mediated decrease in Fn but not the dose‐dependent reduction in the CO2 bolus response. 7. Taken together, these data strongly suggest that the carotid chemoreceptor‐mediated excitation of expiratory bulbospinal neurones is dependent on NMDA but not non‐NMDA glutamate receptors.

Respiratory rhythm generation: converging concepts from in vitro and in vivo approaches?

The timing and activation pattern of breathing movements are determined by the respiratory network. This network is amenable to a variety of in vivo and in vitro approaches, which offers a unique opportunity to investigate multiple organizational levels. It is only recently, however, that concepts obtained under in vivo and in vitro conditions are being integrated into a coherent model of breathing behavior. For example, the pre-Bötzinger complex as an essential site for rhythm generation was first identified in vitro, but has since been verified in vivo. Conversely, timing signals provided by other central and peripheral neuronal areas have so far been investigated in vivo, but it is now possible to address these issues with more complex in vitro preparations. Several key issues remain unresolved. For example, to what extent is the respiratory pattern controlled independently of the underlying rhythm? Answers to this and other questions require a dissection of mechanisms that is only possible through a complementary combination of experimental approaches.

Clinically Relevant Infusion Rates of μ-Opioid Agonist Remifentanil Cause Bradypnea in Decerebrate Dogs but not Via Direct Effects in the pre-Bötzinger Complex Region

Systemic administration of mu-opioids at clinical doses for analgesia typically slows respiratory rate. Mu-opioid receptors (MORs) on pre-Bötzinger Complex (pre-BötC) respiratory neurons, the putative kernel of respiratory rhythmogenesis, are potential targets. The purpose of this study was to determine the contribution of pre-BötC MORs to the bradypnea produced in vivo by intravenous administration of clinically relevant infusion rates of remifentanil (remi), a short-acting, potent mu-opioid analgesic. In decerebrate dogs, multibarrel micropipettes were used to record pre-BötC neuronal activity and to eject the opioid antagonist naloxone (NAL, 0.5 mM), the glutamate agonist D-homocysteic acid (DLH, 20 mM), or the MOR agonist [D-Ala(2), N-Me-Phe(4), gly-ol(5)]-enkephalin (DAMGO, 100 microM). Inspiratory and expiratory durations (T(I) and T(E)) and peak phrenic nerve activity (PPA) were measured from the phrenic neurogram. The pre-BötC was functionally identified by its rate altering response (typically tachypnea) to DLH microinjection. During intravenous remi-induced bradypnea (approximately 60% decrease in central breathing frequency, f(B)), bilateral injections of NAL in the pre-BötC did not change T(I), T(E), f(B), and PPA. Also, NAL picoejected onto single pre-BötC neurons depressed by intravenous remi had no effect on their discharge. In contrast, approximately 60 microg/kg of intravenous NAL rapidly reversed all remi-induced effects. In a separate group of dogs, microinjections of DAMGO in the pre-BötC increased f(B) by 44%, while subsequent intravenous remi infusion more than offset this DAMGO induced tachypnea. These results indicate that mu-opioids at plasma concentrations that cause profound analgesia produce their bradypneic effect via MORs located outside the pre-BötC region.

Dose-dependent Effects of Halothane on the Carbon Dioxide Responses of Expiratory and Inspiratory Bulbospinal Neurons and the Phrenic Nerve Activities in Dogs

Background:Expiratory bulbospinal and inspiratory bulbospinal neurons in the ventral respiratory group provide drive for thoracoabdominal expiratory and phrenic and thoracic inspiratory motor neurons. Potent inhalational agents such as halothane may have differential effects on inspiratory and expiratory neurons, but detailed studies comparing neurons at a homologous level are lacking. Methods:The dose-dependent effects of anesthesia with 1.0- 2.5 minimum alveolar concentration halothane on the CO2 responses of single expiratory and inspiratory bulbospinal neurons of the ventral respiratory group and on phrenic neural activities were studied in nonpremedicated, anesthetized, paralyzed, vagotomlzed dogs. Hyperventllation with O2 and the addition of CO2-O2 mixtures were used to produce low, medium, and high steady-state levels of central CO2 drive. Results:Peak neuron discharge frequency decreased progressively with increasing halothane dose at all levels of CO2 drive for both types of neurons. The sensitivities of inspiratory and expiratory bulbospinal neuronal activities to halothane were not significantly different from one another, whereas the sensitivity to halothane of the peak phrenic activity was markedly greater than those of the neurons. Increasing halothane dose caused a downward, predominantly parallel shift of the CO2 response curves. Phrenic nerve activity also showed a decrease in slope of the CO2 response. Conclusions:The activities of respiratory premotor neurons are less depressed by Increasing doses of halothane than is phrenic nerve activity. The greater depression of phrenic activity may result from additional anesthetic actions on the efferent motor pathways, resulting in decreased descending synaptic inputs to phrenic motor neurons.

Systemic Arterial Blood pH Servocontrol of Mechanical Ventilation

Servocontrol of mechanical ventilation using systemic arterial blood pH, measured by a dual-function pH/PCO2 intra-arterial sensor, as the controlled variable uas carried out in 30 dogs anesthetized with pentobarbital, 30 mg/kg. The control loop consisted of the animal, an intra-arterial dual-function pH/PCO2 sensor and sensor amplifier, a controller, and a Siemans-Elema 900 servoventilator. The system responded appropriately to changes in set-point pH from 7.30 to 7.50, as well as to infusions of lactic acid, which, with the control loop open, decreased systemic arterial blood pH 0.1 TO 0.2 PH units. Long-term (16 hr) ventilation of one dog with the systemic arterial blood pH servocontrol ventilator was shown to be feasible.