Subtype composition and responses of respiratory neurons in the pre-botzinger region to pulmonary afferent inputs in dogs.

The brain stem pre-Botzinger complex (pre-BC) plays an important role in respiratory rhythm generation. However, it is not clear what function each subpopulation of neurons in the pre-BC serves. The purpose of the present studies was to identify neuronal subpopulations of the canine pre-BC and to characterize the neuronal responses of subpopulations to experimentally imposed changes in inspiratory (I) and expiratory (E) phase durations. Lung inflations and electrical stimulation of the cervical vagus nerve were used to produce changes in respiratory phase timing via the Hering-Breuer reflex. Multibarrel micropipettes were used to record neuronal activity and for pressure microejection in decerebrate, paralyzed, ventilated dogs. The pre-BC region was functionally identified by eliciting tachypneic phrenic neural responses to localized microejections of DL-homocysteic acid. Antidromic stimulation and spike-triggered averaging techniques were used to identify bulbospinal and cranial motoneurons, respectively. The results indicate that the canine pre-BC region consists of a heterogeneous mixture of propriobulbar I and E neuron subpopulations. The neuronal responses to ipsi-, contra-, and bilateral pulmonary afferent inputs indicated that I and E neurons with decrementing patterns were the only neurons with responses consistently related to phase duration. Late-I neurons were excited, but most other types of I neurons were inhibited or unresponsive. E neurons with augmenting or parabolic discharge patters were inhibited by ipsilateral inputs but excited by contra- and bilateral inputs. Late-E neurons were more frequently encountered and were inhibited by ipsi- and bilateral inputs, but excited by contralateral inputs. The results suggest that only a limited number of neuron subpopulations may be involved in rhythmogenesis, whereas many neuron types may be involved in motor pattern generation.

Differential modulation of respiratory neuronal discharge patterns by GABA(A) receptor and apamin-sensitive K(+) channel antagonism.

The discharge patterns of respiratory neurons of the caudal ventral respiratory group (cVRG) appear to be subject to potent GABAergic gain modulation. Local application of the GABA(A) receptor antagonist bicuculline methochloride amplifies the underlying discharge frequency (F(n)) patterns mediated by endogenous excitatory and inhibitory synaptic inputs. Gain modulation can also be produced by alterations in the amplitude of spike afterhyperpolarizations (AHPs) mediated by apamin-sensitive small-conductance Ca(2+)-activated K(+) (SK) channels. Since methyl derivatives of bicuculline (BICm) also have been shown to reduce the amplitude of AHPs, in vitro, it is possible that the BICm-induced gain modulation is due to a block of SK channels. The purpose of these studies was to determine the mechanisms by which BICm produces gain modulation and to characterize the influence of SK channels in the control of respiratory neuron discharge. Six protocols were used in this in vivo study of cVRG inspiratory (I) and expiratory (E) neurons in decerebrate, paralyzed, ventilated dogs. The protocols included characterizations of the neuronal responses to 1) BICm and apamin on the same neuron, 2) BICm during maximum apamin-induced block of AHPs, 3) apamin during maximum BICm-induced gain modulatory responses, 4) the specific GABA(A) receptor antagonist, (+)beta-hydrastine, 5) the specific GABA(A) receptor agonist, muscimol, and 6) the GABA uptake inhibitor, nipecotic acid. For protocols 3, 5, and 6, only E neurons were studied. Four-barrel micropipettes were used for extracellular single neuron recording and pressure ejection of drugs. Cycle-triggered histograms were used to quantify the F(n) patterns and to determine the drug-induced changes in the gain (slope) and offset of the F(n) patterns. Compared to apamin at maximum effective dose rates, BICm produced a 2.1-fold greater increase in peak F(n) and a 3.1-fold greater increase in average F(n). BICm and apamin produced similar increases in gain, but the offsets due to apamin were more negative. The responses to hydrastine were similar to BICm. During maximum apamin block, BICm produced an additional 112 +/- 22% increase in peak F(n). Conversely, apamin produced an additional 176 +/- 74% increase in peak F(n) during the maximum BICm-induced response. Muscimol and nipecotic acid both decreased the gain and offset of the discharge patterns. Taken together, these results suggest that the gain modulatory effect of BICm is due to a reduction of GABA(A)-ergic shunting inhibition rather than a reduction in AHPs by block of SK channels in canine cVRG neurons.

Relative magnitude of tonic and phasic synaptic excitation of medullary inspiratory neurons in dogs.

The relative contribution of phasic and tonic excitatory synaptic drives to the augmenting discharge patterns of inspiratory (I) neurons within the ventral respiratory group (VRG) was studied in anesthetized, ventilated, paralyzed, and vagotomized dogs. Multibarrel micropipettes were used to record simultaneously single-unit neuronal activity and pressure microejected antagonists of GABAergic, glycinergic, N-methyl-D-aspartate (NMDA) and non-NMDA glutamatergic, and cholinergic receptors. The discharge patterns were quantified via cycle-trigger histograms. The findings suggest that two-thirds of the excitatory drive to caudal VRG I neurons is tonic and mediated by NMDA receptors and the other third is ramp-like phasic and mediated by non-NMDA receptors. Cholinergic receptors do not appear to be involved. The silent expiratory phase is produced by phasic inhibition of the tonic activity, and approximately 80% of this inhibition is mediated by gamma-aminobutyric acid receptors (GABA(A)) and approximately 20% by glycine receptors. Phasic I inhibition by the I decrementing neurons does not appear to contribute to the predominantly step-ramp patterns of these I neurons. However, this decrementing inhibition may be very prominent in controlling the rate of augmentation in late-onset I neurons and those with ramp patterns lacking the step component.

Effects of halothane on excitatory neurotransmission to medullary expiratory neurons in a decerebrate dog model.

BACKGROUND: The activity of canine expiratory (E) neurons in the caudal ventral respiratory group is primarily dependent on N-methyl-D-aspartic acid (NMDA) receptor-mediated excitatory chemodrive inputs and modulated by an inhibitory mechanism mediated via gamma-aminobutyric acidA (GABA(A)) receptors. In an intact canine preparation, halothane depressed the activity of these neurons mainly by reduction in overall glutamatergic excitation. A new decerebrate preparation allows comparison of the effects of halothane on these synaptic mechanisms with an anesthetic-free baseline state. METHODS: Two separate studies were performed in decerebrate, vagotomized, paralyzed, mechanically ventilated dogs during hypercapnic hyperoxia. In study 1, the effect of 1 minimum alveolar concentration (MAC) halothane on extracellularly recorded E neuronal activity was studied before and during complete GABA(A) receptor blockade by localized pressure ejection of bicuculline. Complete blockade of the inhibitory mechanism allowed differentiation between the effects of halothane on overall GABA(A)-mediated inhibition and on overall NMDA receptor-mediated excitation. In study 2, the effect of 1 MAC halothane on the dose response of neurons to localized picoejection of the glutamate agonist NMDA was used to estimate halothane effect on postsynaptic glutamatergic excitatory neurotransmission. RESULTS: In study 1, the spontaneous activity of 14 E neurons was depressed 38.6 +/- 20.6% (mean +/- SD) by 1 MAC halothane. Overall excitation was depressed 31.5 +/- 15.5%. The GABAergic inhibition showed a 11.7 +/- 18.3% enhancement during halothane. In study 2, the spontaneous activity of 13 E neurons was again significantly depressed by 1 MAC halothane (27.9 +/- 10.6%), but the postsynaptic response of the neurons to exogenous NMDA was not significantly depressed by halothane (3.3 +/- 38.4%). CONCLUSIONS: Together these results suggest that in our E neuron paradigm, halothane exerted its depressive effect mainly via reduction of glutamatergic presynaptic mechanisms.

Effects of halothane on synaptic neurotransmission to medullary expiratory neurons in the ventral respiratory group of dogs.

BACKGROUND: The activity of canine expiratory neurons is primarily dependent on N-methyl-D-aspartic acid (NMDA)-receptor mediated excitatory chemodrive inputs and a powerful inhibitory gain modulatory mechanism mediated via gamma-aminobutyric acidA (GABA(A)) receptors. We examined whether the depressant effect of halothane on expiratory neuronal activity is primarily caused by a reduction in glutamatergic excitation or a potentiation of the inhibitory mechanism. METHODS: Experiments were performed in halothane-anesthetized, vagotomized, paralyzed, and mechanically ventilated dogs during hypercapnic hyperoxia. The effect of a halothane dose increase from one minimum alveolar concentration (MAC) to 2 MAC on extracellularly recorded expiratory neuronal activity was studied before and during complete GABA(A) receptor blockade by localized picoejection of bicuculline close to the neuron. Complete blockade of the inhibitory mechanism allowed differentiation between the effects of halothane on overall NMDA-mediated excitation and on GABA(A)-mediated inhibition. RESULTS: The spontaneous activity of 12 expiratory neurons was significantly depressed (18.1%) by the 1-MAC halothane dose increase. Overall glutamatergic excitation was depressed 38.3+/-12.3% (mean +/- SD) by the 1-MAC halothane increase. The prevailing GABA(A)ergic attenuation of neuronal output decreased significantly from 49.5+/-10 to 32.0+/-10.4%. Thus overall inhibition was reduced by halothane by 33.5+/-17.2%. CONCLUSIONS: These results suggest that the depressive effect of a 1-MAC halothane dose increase on expiratory neuronal activity in our in vivo preparation with an intact neural network was mainly caused by a reduction of synaptic excitatory mechanisms and not an enhancement of synaptic inhibitory mechanisms.

Differential roles of ionotropic glutamate receptors in canine medullary inspiratory neurons of the ventral respiratory group.

The relative roles of ionotropic N-methyl-D-aspartate (NMDA) and non-NMDA glutamate receptors in supplying excitatory drive to inspiratory (I) augmenting pattern neurons of the ventral respiratory group were studied in anesthetized, ventilated, paralyzed, and vagotomized dogs. Multibarrel micropipettes were used to record simultaneously single-unit neuronal activity and pressure microeject the NMDA antagonist, 2-amino-5-phosphonovalerate (AP5; 2 mM), the non-NMDA antagonist 2, 3-dihydroxy-6-nitro-7-sulfamoyl-benzo(f)quinoxaline (NBQX; 0.25 mM), and an artificial cerebrospinal fluid vehicle. Ejected volume-rates were measured directly via meniscus level changes. The moving time average of phrenic nerve activity was used to determine respiratory phase durations and to synchronize cycle-triggered histograms of the discharge patterns. Both AP5 and NBQX produced dose-dependent reductions in peak spontaneous I neuronal discharge frequency (Fn). The average (+/- SE) maximum reduction in peak Fn produced by AP5 was 69.1 +/- 4.2% and by NBQX was 47.1 +/- 3.3%. Blockade of both glutamate receptor subtypes nearly silenced these neurons, suggesting that their activity is highly dependent on excitatory synaptic drive mediated by ionotropic glutamate receptors. Differential effects were found for the two glutamatergic antagonists. AP5 produced downward, parallel shifts in the augmenting pattern of discharge, whereas NBQX reduced the slope of the augmenting discharge pattern. These results suggest that time-varying excitatory input patterns to the canine I bulbospinal neurons are mediated by non-NMDA glutamate receptors and that constant or tonic input patterns to these neurons are mediated by NMDA receptors.

Fibronectin expression in the developing human spinal cord, nerves, and ganglia.

AIM: Analysis of developmental role of fibronectin during differentiation of the human spinal cord, nerves, and ganglia. METHODS: Seven normal human embryos and fetuses between the 7th and 9th developmental week and a 9-week fetus with cervical spina bifida were histologically examined on hematoxylin and eosin stained serial paraffin sections of thoracic axial segments. Monoclonal antibody to the human cell fibronectin fragment was used for immunohistochemical detection of fibronectin. RESULTS: In the 7th and 8th week of development, fibronectin was weakly expressed in the ventricular and intermediate zones of the spinal cord. Intense fibrillar expression was found in the marginal zone of the spinal cord - first over the ventral gray horns and later over the lateral and dorsal gray horns, and along the pathways of ventral and dorsal roots of the spinal nerves and in the spinal ganglia. At 9th week, fibronectin expression disappeared in the ventricular and intermediate zones a nd became weak and granular in the marginal zone of the spinal cord. In the spinal cord of a 9-week malformed fetus with cervical spina bifida, fibronectin expression was completely absent. Fibronectin was expressed in the nerves and ganglia throughout the investigated period, both in normal and malformed human conceptuses. CONCLUSION: Transient expression of fibronectin in the human spinal cord coincided with the most intense neuronal differentiation. Temporal and spatial expression of fibronectin during normal development, and its absence in a malformed human fetus suggests developmental role of fibronectin for the normal formation of the spinal cord.

Differential effects of GABAA receptor antagonists in the control of respiratory neuronal discharge patterns.

To ascertain the role of the inhibitory neurotransmitter gamma-aminobutyric acid (GABA) in shaping and controlling the phasic discharge patterns of medullary respiratory premotor neurons, localized pressure applications of the competitive GABAA receptor antagonist bicuculline (BIC) and the noncompetitive GABAA receptor antagonist picrotoxin (PIC) were studied. Multibarrel micropipettes were used in halothane anesthetized, paralyzed, ventilated, vagotomized dogs to record single unit activity from inspiratory and expiratory neurons in the caudal ventral respiratory group and to picoeject GABAA receptor antagonists. The moving time average of phrenic nerve activity was used to determine respiratory phase durations and to synchronize cycle-triggered histograms of discharge patterns. Picoejection of BIC and PIC had qualitatively different effects on the discharge patterns of respiratory neurons. BIC caused an increase in the discharge rate during the neuron's active phase without inducing activity during the neuron's normally silent phase. The resulting discharge patterns were amplified replicas (x2-3) of the underlying preejection phasic patterns. In contrast, picoejection of PIC did not increase the peak discharge rate during the neuron's active phase but induced a tonic level of activity during the neuron's normally silent phase. The maximum effective BIC dose (15 +/- 1.8 pmol/min) was considerably smaller than that for PIC (280 +/- 53 pmol/min). These findings suggest that GABAA receptors with differential pharmacology mediate distinct functions within the same neuron, 1) gain modulation that is BIC sensitive but PIC insensitive and 2) silent-phase inhibition blocked by PIC. These studies also suggest that the choice of an antagonist is an important consideration in the determination of GABA receptor function within the respiratory motor control system.

Improved method of canine decerebration.

We describe an improved decerebration method for dogs that is suitable for studies of brain stem neurons in the absence of anesthesia. Previously reported techniques of canine decerebration often lead to respiratory and hemodynamic instability and lack of typical decerebrate rigidity. We have developed a precise, visually controlled, midcollicular brain stem transection technique that overcomes these problems. Our method results in only moderate blood loss while preserving carotid and basilar artery circulations. Consistent levels of brain stem transection routinely lead to stable postdecerebration hemodynamic parameters, allowing prolonged brain stem neuronal recordings. The same model should also be useful for a variety of studies involving other physiological systems in dogs in the absence of anesthesia and for studies of anesthetic effects.

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.

Dose-dependent effects of halothane on the phrenic nerve responses to acute hypoxia in vagotomized dogs.

BACKGROUND: Previous studies in dogs and humans suggest that the carotid body chemoreceptor response to hypoxia is selectively impaired by halothane. The present studies in an open-loop canine preparation were performed to better delineate the effects of anesthetic concentrations of halothane on the carotid body chemoreceptor-mediated phrenic nerve response to an acute hypoxic stimulus. METHODS: Three protocols were performed to study the effects of halothane anesthesia on the phrenic nerve response to 1 min of isocapnic hypoxia (partial pressure of oxygen [PaO2] at peak hypoxia, 35-38 mmHg) in unpremedicated, anesthetized, paralyzed, vagotomized dogs during constant mechanical ventilation. In protocol 1, the dose-dependent effects of halothane from 0.5-2.0 minimum alveolar concentration (MAC) on the hypoxic response during moderate hypercapnia (partial pressure of carbon dioxide [PaCO2], 60-65 mmHg) were studied in 10 animals. In protocol 2, the hypoxic responses at 1 MAC halothane near normocapnia (PaCO2, 40-45 mmHg) and during moderate hypercapnia were compared in an additional four animals. In protocol 3, the hypoxic response of 4 of 10 dogs from protocol 1 was also studied under sodium thiopental (STP) anesthesia after they completed protocol 1. RESULTS: Protocol 1: Peak phrenic nerve activity (PPA) increased significantly during the hypoxic runs compared with the isocapnic hyperoxic controls at all halothane doses. The phrenic nerve response to the hypoxic stimulus was present even at the 2 MAC dose. Protocol 2: The net hypoxic responses for the two carbon dioxide background levels at 1 MAC were not significantly different. Protocol 3: The net hypoxic response of PPA for the STP anesthetic was not significantly different from the 1 MAC halothane dose. Bilateral carotid sinus denervation abolished the PPA response to hypoxia. CONCLUSIONS: The phrenic nerve response to an acute, moderately severe isocapnic hypoxic stimulus is dose-dependently depressed but not abolished by surgical doses of halothane. This analysis does not suggest a selective depression of the carotid body chemoreceptor response by halothane. The observed hypoxic phrenic response was mediated by the carotid body chemoreceptors in vagotomized dogs because bilateral carotid sinus denervation abolished all increases in PPA.

Effects of halothane on the phrenic nerve responses to carbon dioxide mediated by carotid body chemoreceptors in vagotomized dogs.

BACKGROUND: Previous studies in dogs showed that the phrenic nerve response to an acute hypoxic stimulus was dose dependently depressed by 0.5-2.0 minimum alveolar concentration (MAC) of halothane but not abolished. Because a carbon dioxide stimulus is transduced by a different mechanism in the carotid body chemoreceptors (CBCRs) than is a hypoxic stimulus, inhalational anesthetics may preferentially depress one of these transduction processes, the central neuronal processing, or both, of the integrated responses to these two types of inputs. METHODS: Carotid body chemoreceptor stimulation was produced by short (1-1.5 s), bilateral, 100% carbon dioxide in saline infusions into the carotid arteries during neural inspiration in unpremedicated, halothane-anesthetized, paralyzed, vagotomized dogs during constant mechanical ventilation. The phrenic neurogram quantified the neural inspiratory response. Four protocols were performed in the study: (1) the dose-dependent effects of halothane anesthesia (0.5-2.0 MAC) during hyperoxic hypercapnia on phrenic nerve activity, (2) the effects of three background levels of the partial pressure of carbon dioxide (PaCO2) on the magnitude of the carbon dioxide infusion responses at 1 MAC halothane, (3) the effects of anesthetic type on the magnitude of the carbon dioxide infusion response, and (4) the effects of CBCR denervation. RESULTS: Peak phrenic nerve activity (PPA) increased significantly during the carbon dioxide-stimulated phrenic burst in protocols 1-3; after denervation there was no response (protocol 4). Halothane produced a dose-dependent reduction in the PPA of control and carbon dioxide infusion-stimulated phrenic bursts and in the net carbon dioxide response. The net PPA responses for the different PaCO2 background levels were not different but were somewhat larger for sodium thiopental anesthesia than for 1.0 MAC halothane. CONCLUSIONS: The phrenic nerve response to an acute, severe carbon dioxide stimulus was dose dependently depressed by surgical doses of halothane. The observed responses to carbon dioxide infusion were mediated by the CBCRs because they were eliminated by CBCR denervation. These results suggest that the CBCR transduction and central transmission of the carbon dioxide signal in terms of inspiratory excitatory drive are not abolished at surgical levels of halothane anesthesia.

Differences in origin and fate between the cranial and caudal spinal cord during normal and disturbed human development.

Differences in histological appearance between the cranial and caudal parts of the spinal cord and associated axial organs were analyzed in 9- and 15-week-old human dysraphic fetuses and compared with normal fetuses. In human development the cranial part of the neural tube down to the lumbosacral level forms during primary neurulation, while its caudal part results from secondary neurulation. In the 9-week fetus with cervical spina bifida, the cranial spinal cord displayed a variety of morphological changes along the cranio-caudal axis. Spinal cord in the upper cervical region transformed into the area cerebrovasculosa, while the lower cervical and thoracic levels showed only disturbed differentiation of the cell layers and roof plate. The degree of the cranial spinal cord dysmorphogenesis correlated with anomalies of the underlying notochord and vertebral column. The caudal to lumbosacral region of the spinal cord appeared normal. In the case of the 15-week-old fetus with complete dysraphia, the area cerebrovasculosa was found along the whole extent of the cranial spinal cord, while more caudally, all axial organs showed a normal histological structure. Our findings confirmed a different origin for the cranial and caudal parts of the human spinal cord. The appearance of dysraphic disorders corresponded to the time of primary neurulation; therefore, they resulted in the faulty formation of the cranial spinal cord. Normally formed caudal spinal cord appears during secondary neurulation at later developmental stages.