Membrane Potential Trajectories Research Articles

Glutamic acid decarboxylase-immunoreactivity of bulbar respiratory neurons identified by intracellular recording and labeling in rats

To distinguish the GABAergic neuron in the ventral respiratory group (VRG) of rats, immunohistochemical staining of glutamic acid decarboxylase (GAD) was performed in neurons that had been individually identified by in vivo intracellular recording and labeling with neurobiotin. A total of five types of respiratory neurons were identified and labeled; augmenting inspiratory (aug-I, n=12), decrementing or early inspiratory (early-I, n=3), inspiration–expiration phase spanning or late inspiratory (late-I, n=3), decrementing expiratory or postinspiratory (PI, n=8), and augmenting or stage 2 expiratory (E2, n=3). In addition, expiration–inspiration phase-spanning or pre-inspiratory neurons (pre-I, n=2) were recorded, but not labeled. The membrane potential trajectory of each neuron type resembled that previously described in cat, suggesting a comparable neuronal organization between the two species. According to the axonal arborization, those labeled neurons were further classified as propriobulbar (6 aug-I, all early-I, all late-I, and 3 PI), bulbospinal (2 aug-I and all E2) and cranial-motor neurons (4 aug-I and 5 PI). GAD-immunoreactivity was consistently detected in the propriobulbar neurons, while it was not seen in cranial-motor and bulbospinal neurons. In addition, GAD-immunoreactive varicosities were found surrounding the somatic and dendritic surface of all labeled neurons. The present results illustrate that the propriobulbar types of early-I, aug-I, late-I and PI neurons are GABAergic inhibitory neurons and virtually all types of respiratory neurons receive GABAergic inputs in the rat’s VRG.

Simulation of cross-correlograms resulting from synaptic connections between neurons

A cross-correlation simulation program is presented. It is intended for use as a rapid initial screening test, to discriminate between possible connection configurations that may underlie observed cross-correlations. It can also serve to introduce students to the event cross-correlation technique. The simulation employs a simple model of two neurons with noisy membranes that may excite or inhibit one another or receive a common input from a third neuron. The model differs from its predecessors in that the user specifies the neurons and their interconnections by the resulting membrane potential trajectories, rather than discharge statistics. Illustrations of some simple performance characteristics are given, as well as examples of cross-correlograms resulting from more complex interactions between neurons. The latter demonstrate the usefulness of the simulation in the interpretation of cross-correlograms obtained from experimental data. The simulation is a LabVIEW (National Instruments) program executable under the Windows operating system (Microsoft) and is available for downloading from website www.utoronto.ca/respgrp/sim.htm.

Models of respiratory rhythm generation in the pre-Bötzinger complex. I. Bursting pacemaker neurons.

A network of oscillatory bursting neurons with excitatory coupling is hypothesized to define the primary kernel for respiratory rhythm generation in the pre-Bötzinger complex (pre-BötC) in mammals. Two minimal models of these neurons are proposed. In model 1, bursting arises via fast activation and slow inactivation of a persistent Na+ current INaP-h. In model 2, bursting arises via a fast-activating persistent Na+ current INaP and slow activation of a K+ current IKS. In both models, action potentials are generated via fast Na+ and K+ currents. The two models have few differences in parameters to facilitate a rigorous comparison of the two different burst-generating mechanisms. Both models are consistent with many of the dynamic features of electrophysiological recordings from pre-BötC oscillatory bursting neurons in vitro, including voltage-dependent activity modes (silence, bursting, and beating), a voltage-dependent burst frequency that can vary from 0.05 to >1 Hz, and a decaying spike frequency during bursting. These results are robust and persist across a wide range of parameter values for both models. However, the dynamics of model 1 are more consistent with experimental data in that the burst duration decreases as the baseline membrane potential is depolarized and the model has a relatively flat membrane potential trajectory during the interburst interval. We propose several experimental tests to demonstrate the validity of either model and to differentiate between the two mechanisms.

Multifunctional Laryngeal Motoneurons: an Intracellular Study in the Cat

We studied the patterns of membrane potential changes in laryngeal motoneurons (LMs) during vocalization, coughing, swallowing, sneezing, and the aspiration reflex in decerebrate paralyzed cats. LMs, identified by antidromic activation from the recurrent laryngeal nerve, were expiratory (ELMs) or inspiratory (ILMs) cells that depolarized during their respective phases in eupnea. During vocalization, most ELMs depolarized and most ILMs hyperpolarized. Some ILMs depolarized slightly during vocalization. During coughing, ELMs depolarized abruptly at the transition from the inspiratory to the expiratory phase. In one-third of ELMs, this depolarization persisted throughout the abdominal burst. In the remainder ("type A"), it was interrupted by a transient repolarization. ILMs exhibited a membrane potential trajectory opposite to that of type A ELMs during coughing. During swallowing, the membrane potential of ELMs decreased transiently at the onset of the hypoglossal burst and then depolarized strongly during the burst. ILMs hyperpolarized sharply at the onset of the burst and depolarized as hypoglossal activity ceased. During sneezing, ELMs and ILMs exhibited membrane potential changes similar to those of type A ELMs and ILMs during coughing. During the aspiration reflex, ELMs and ILMs exhibited bell-shaped hyperpolarization and depolarization trajectories, respectively. We conclude that central drives to LMs, consisting of complex combinations of excitation and inhibition, vary during vocalization and upper airway defensive reflexes. This study provides data for analysis of the neuronal networks that produce these various behaviors and analysis of network reorganization caused by changes in dynamic connections between the respiratory and nonrespiratory neuronal networks.

A model of signal processing at a mammalian sympathetic neurone

A computational model has been developed for the action potential and, more generally, the electrical behaviour of the rat sympathetic neurone. The neurone is simulated as a complex system in which five voltage-dependent conductances ( g Na, g Ca, g KV, g A, g KCa), one Ca 2+-dependent voltage-independent conductance ( g AHP) and the activating synaptic conductance coexist. The individual currents are mathematically described, based on a systematic analysis obtained for the first time in a mature and intact mammalian neurone using two-electrode voltage-clamp experiments. The simulation initiates by setting the starting values of each variable and by evaluating the holding current required to maintain the imposed membrane potential level. It is then possible to simulate current injection to reproduce either the experimental direct stimulation of the neurone or the physiological activation by the synaptic current flow. The subthreshold behaviour and the spiking activity, even during long-lasting current application, can be analysed. At every time step, the program calculates the amplitude of the individual currents and the ensuing changes; it also takes into account the accompanying K + accumulation process in the perineuronal space and changes in Ca 2+ load. It is shown that the computed time course of membrane potential must be filtered, in order to reproduce the limited bandwidth of the recording instruments, if it is to be compared with experimental measurements under current-clamp conditions. The membrane potential trajectory and single current data are written in files readable by graphic software. Finally, a screen image is obtained which displays in separate graphs the membrane potential time course, the synaptic current and the six ionic current flows. The simulated action potentials are comparable to the experimental ones as concerns overshoot amplitude and rising and falling rates. Therefore, this program is potentially helpful in investigating many aspects of neurone behaviour.

Synaptic connections to phrenic motoneurons in the decerebrate rat.

Phrenic motoneuron membrane potential trajectories in decerebrate rats exhibit three stages; depolarisation during inspiration, a decreased depolarisation during early expiration and hyperpolarization during late expiration. These trajectories are a result of excitation by ventral-group medullary inspiratory neurons and upper-cervical inspiratory neurons during inspiration and the early part of expiration, and inhibition from Bötzinger-complex expiratory neurons during the late part of expiration.

Synchronization of ventral-group, bulbospinal inspiratory neurons in the decerebrate rat.

We examined the synaptic connections between ventral-group, bulbospinal inspiratory neurons in 27 vagotomized, paralyzed, ventilated, and decerebrated rats using cross-correlation and spike-triggered averaging of intracellular potentials. The neurons were recorded in the medulla about the level of the obex and identified by their inspiratory firing pattern and antidromic activation from the spinal cord at C7. Whole C5 phrenic nerve recordings were made using bipolar electrodes from the central cut ends of the nerve. Most (108/137, 79%) inspiratory neurons discharged only during inspiration but some (29/137, 21%) also discharged during early expiration. Their intracellular membrane potentials displayed a pattern of depolarization during inspiration, repolarization during early expiration, and hyperpolarization during late expiration. Intracellular chloride iontophoresis changed the inspiratory membrane potential trajectories from augmenting to decrementing in 11 of 19 neurons tested (58%), and demonstrated the presence of both early-decrementing and late-augmenting waves of inhibitory postsynaptic potentials during expiration in 11 of 19 neurons tested (58%). Cross-correlation histograms were computed between pairs of extracellularly recorded neurons to detect short time scale synchronizations indicative of synaptic connections (26 ipsilateral; 23 contralateral). While none of the cross-correlation histograms for contralateral pairs showed peaks, most (23, 88%) of those for ipsilateral pairs showed peaks (mean half-amplitude width +/- SD = 1.3 +/- 0.4 ms) at time zero suggestive of common activation. Some of the latter (6, 23%) showed troughs superimposed on the central peaks (mean half-amplitude width +/- SD = 0.9 +/- 0.2 ms) at short latencies (mean latency +/- SD = 1.8 +/- 1.9 ms) suggestive of inhibition; others (8, 31%) had asymmetrical central peaks and two had bilateral peaks suggesting more complex interconnections. Averages of intracellular membrane potentials of inspiratory neurons (n = 24), triggered by action potentials of a nearby extracellularly recorded inspiratory neuron, were computed to detect synchronized postsynaptic potentials. Over half (16, 67%) showed postsynaptic potentials (mean amplitude +/- SD = 201 +/- 176 microV; mean half-amplitude width +/- SD = 2.3 +/- 0.8 ms) confirming the cross-correlation findings of common excitation. We conclude that in decerebrated rats, ventral-group inspiratory neurons projecting to the C7 spinal segment share powerful, ipsilaterally distributed excitatory inputs which enhance their synchronous activity during inspiration. They also receive inhibition during inspiration and early-decrementing and late-augmenting inhibitory inputs during expiration.

Respiratory activity in the facial nucleus in an in vitro brainstem of tadpole, Rana catesbeiana.

1. In studies of the central neural control of breathing, little advantage has been taken of comparative approaches. We have developed an in vitro brainstem preparation using larval Rana catesbeiana which generates two rhythmic neural activities characteristic of lung and gill ventilation. Based on the pattern of the facial (VII) nerve activity both lung and gill rhythm-related respiratory cycles were divided into three distinct phases. The purpose of this study was to characterize and classify membrane potential trajectories of respiratory motoneurons in the VII nucleus at intermediate stages (XII-XVII) of development. 2. Seventy-five respiratory-modulated neurons were recorded intracellularly within the facial motor nucleus region. Their resting membrane potential was between -40 and -80 mV. Sixty of them were identified as VII motoneurons and fifteen were non-antidromically activated. Membrane potentials of fifty-six of the seventy-five neurons were modulated with both lung (5-27 mV) and gill rhythms (3-15 mV) and the remaining nineteen neurons had only a modulation with lung rhythmicity (6-23 mV). No cells with gill modulation alone were observed. 3. All of the cells modulated with lung rhythmicity had only phase-bound depolarizing or hyperpolarizing membrane potential swings which could be categorized into four distinct patterns. In contrast, of the fifty-six cells modulated with gill rhythmicity, thirty-two were phasically depolarized during distinct phases of the gill cycle (four patterns were distinguished), whereas the remaining twenty-four were phase spanning with two distinct patterns. The magnitudes of lung and gill modulations were proportionally related to each other in the cells modulated with both rhythms. 4. In all sixteen neurons studied, a reduction or a reversal of phasic inhibitory inputs during a portion of the lung or gill respiratory cycle was observed following a negative current or chloride ion (Cl-) injection. The phasic membrane resistance modulation in relation to the gill rhythm was analysed in six neurons and a relative decrease in the somatic membrane resistance (0.7-8.1 M omega) was detected during the periods of hyperpolarization. 5. We propose that, at these intermediate stages of development: (a) both gill and lung respiratory oscillations in motoneurons are generated by respiratory premotor neurons having only a few distinct activity patterns; (b) these patterns delineate distinct portions of the centrally generated respiratory cycles; and (c) phasic synaptic inhibition, mediated by Cl-, contributes to shaping the membrane potential trajectories of respiratory motoneurons.

Electroresponsive properties and membrane potential trajectories of three types of inspiratory neurons in the newborn mouse brain stem in vitro.

1. The electrophysiological properties of inspiratory neurons were studied in a rhythmically active thick-slice preparation of the newborn mouse brain stem maintained in vitro. Whole cell patch recordings were performed from 60 inspiratory neurons within the rostral ventrolateral part of the slice with the aim of extending the classification of inspiratory neurons to include analysis of active membrane properties. 2. The slice generated a regular rhythmic motor output recorded as burst of action potentials on a XII nerve root with a peak to peak time of 11.5 +/- 3.4 s and a duration of 483 +/- 54 ms (means +/- SD, n = 50). Based on the electroresponsive properties and membrane potential trajectories throughout the respiratory cycle, three types of inspiratory neurons could be distinguished. 3. Type-1 neurons were spiking in the interval between the inspiratory potentials (n = 9) or silent with a resting membrane potential of -48.6 +/- 10.1 mV and an input resistance of 306 +/- 130 M omega (n = 15). The spike activity between the inspiratory potentials was burst-like with spikes riding on top of an underlying depolarization (n = 11) or regular with no evidence of bursting (n = 12). Hyperpolarization of the neurons below threshold for spike initiation did not reveal any underlying phasic synaptic activity, that could explain the bursting behavior. 4. Type-1 neurons showed delayed excitation after hyperpolarizing square current pulses or when the neurons were depolarized from a hyperpolarized level. This membrane behavior resembles the response seen in other CNS neurons expressing an IA. The response to 1-s long depolarizing pulses with a large current strength showed signs of activation of an active depolarizing membrane response leading to a transient reduction in the spike amplitude. The relationship between the membrane potential and the amplitude of square current pulses (Vm-I) showed a small upward rectification below -70 mV, and spike adaptation throughout a 1-s pulse had a largely linear time course. 5. Type-1 neurons depolarized and started to fire spikes 398 +/- 102 ms (n = 20) before the upstroke of the integrated XII nerve discharge. The inspiratory potential was followed by fast hyperpolarization, a short fast-repolarizing phase (1,040 +/- 102 ms, n = 5) and a longer slow-repolarizing phase (lasting until the next inspiratory discharge). 6. Type-2 neurons were spiking in the interval between the inspiratory potentials with no evidence of bursting behavior and had an input resistance of 296 +/- 212 M omega (n = 26). The response to hyperpolarizing pulses revealed an initial sag and postinhibitory rebound depolarization. This membrane behavior resembles the response seen in other CNS neurons expressing an Ih. The Vm-I relationship was linear at depolarized potentials and showed a marked upward rectification below -60 mV. Spike trains elicited by 1-s long pulses showed a pronounced early and late adaptation. 7. Type-2 neurons depolarized and started to fire spikes 171 +/- 87 ms (n = 23) before the upstroke of the integrated XII nerve discharge. The inspiratory potential had a variable amplitude from cell to cell and was followed by a short hyperpolarization in the cells displaying a large amplitude inspiratory potential.(ABSTRACT TRUNCATED AT 250 WORDS)

The shape of the membrane potential trajectory in tonically-active human motoneurons

An innovative technique for estimating the shape of the membrane potential trajectory in voluntarily activated human motoneurons is described. This method involves establishing stimulus-induced interval histograms (SIIH) using the H-reflex algorithm, in which the common peroneal nerve was stimulated and the action potentials of voluntarily-activated low-threshold tibialis anterior motor units were recorded using intramuscular wire electrodes. At least two different stimulus intensities were used for each motor unit and SIIHs for both stimuli were established and compared. It is proposed that the position of the mode of a SIIH (as the abscissa) and the amplitude of a given excitatory postsynaptic potential (EPSP) or its normalized S value (as the ordinate) can be used to estimate the coordinates of a point on the average membrane potential trajectory as it approaches the firing threshold. The new estimated average membrane potential trajectory is at variance with the previously suggested linear trajectory. This new trajectory displayed a ‘slow depolarization’ phase at around 2 mV from the firing threshold. This phase continued for about 20% of the interspike interval (ISI) and was terminated by a rapid depolarization phase, ‘subthreshold rectification’. Using this new trajectory, the previously estimated EPSP and IPSP amplitudes can be reexamined where the trajectories were assumed to approach the firing threshold linearly. The new trajectory may help explain the peak that follows an inhibitory reflex, and the difficulty in grading the stimulus intensity for reflex studies.

Neural organization of the ventilatory activity in the frog, Rana catesbeiana. II

The paralyzed, decerebrate frog, Rana catesbeiana, displays "fictive" oropharyngeal and pulmonary ventilations. In order to evaluate the neuronal correlates of these two centrally programmed ventilatory bursting patterns, we have performed intra- and extracellular recordings of bulbar respiratory neurons in this fictively breathing preparation. A total of 123 respiratory neurons were recorded from the caudal medulla. Of 51 antidromically activated neurons, 20 were vagal motoneurons and 31 were hypoglossal motoneurons. Respiratory neurons that depolarized during the lung (L) or non-lung (N) ventilatory phases were classified as L or N neurons, respectively. Phase spanning neurons (S) were active during both L and N phases. Some neurons showed oscillations of membrane potential synchronous with oropharyngeal ventilation. Those active during the buccal elevation phase were exclusively L neurons, whereas those having buccal depressor activity were exclusively N neurons. Synaptic drive potentials were observed in all neurons recorded intracellularly. In some neurons, hyperpolarization was caused by inhibitory postsynaptic potentials, as demonstrated by reversal of membrane potential trajectory after intracellular chloride iontophoresis. Some individual motoneurons and interneurons exhibited both pulmonary and buccal ventilatory activity, indicating that both pattern generators project to a common motor control system.

Modulation of swimming behavior in the medicinal leech. I. Effects of serotonin on the electrical properties of swim-gating cell 204.

The effects of serotonin on the electrical properties of swim-gating neurons (cell 204) were examined in leech (Hirudo medicinalis) nerve cords. Exposure to serotonin decreased the threshold current required to elicit swim episodes by prolonged depolarization of an individual cell 204 in isolated nerve cords. This effect was correlated with a more rapid depolarization and an increased impulse frequency of cell 204 in the first second of stimulation. In normal leech saline, brief depolarizing current pulses (1 s) injected into cell 204 failed to elicit swim episodes. Following exposure to serotonin, however, identical pulses consistently evoked swim episodes. Thus, serotonin appears to transform cell 204 from a gating to a trigger cell. Serotonin had little effect on the steady-state current-voltage relation of cell 204. However, serotonin altered the membrane potential trajectories in response to injected current pulses and increased the amplitude of rebound responses occurring at the offset of current pulses. These changes suggest that serotonin modulates one or more voltage dependent conductances in cell 204, resulting in a more rapid depolarization and greater firing rate in response to injected currents. Thus, modulation of intrinsic ionic conductances in cell 204 may account in part for the increased probability of swimming behavior induced by serotonin in intact leeches.

A Simple Method for Measurement of Ureteric Peristaltic Function In Vivo and the Effects of Drugs Acting on Ion Channels Applied from the Ureter Lumen in Anesthetized Rats

Effects of isoflurane on the membrane potential trajectory and synaptic activity in bulbar respiratory neurons were investigated in decerebrate, vagotomized and artificially ventilated cats. A 2-min inhalation of 1.6% end-tidal concentration of isoflurane produced depolarization of the membrane in 10 out of 18 inspiratory, 8 out of 15 post-inspiratory and 5 out of 12 expiratory neurons and hyperpolarization in the rest of the population recorded in the ventral respiratory group. In both depolarized and hyperpolarized cells, periodically occurring excitatory and inhibitory synaptic waves were decreased, and input resistance was increased. Concomitantly, isoflurane reduced the excitatory and inhibitory postsynaptic potentials evoked by electrical stimulation of the vagus nerve, superior laryngeal nerve and cervical spinal cord. The effects of isoflurane on membrane potential and input resistance became negligible when excitatory and inhibitory synaptic potentials were suppressed by iontophoretically applied tetrodotoxin. The present results suggest that the respiratory neuronal responses induced by isoflurane are attributed mainly to the decrease of excitatory and inhibitory synaptic interactions in the bulbar respiratory network of neurons.

Variations in membrane potential trajectory of post-inspiratory neurons in the ventrolateral medulla of the cat

In decerebrate cats, intracellular recordings were made in 124 expiratory neurons displaying either a plateau-phase of depolarization during post-inspiration or a steadily decrementing depolarization throughout expiration. Both groups consisted of vagal motoneurons and non-antidromically activated neurons. Five neurons were antidromically activated by both vagal and spinal cord stimuli. The pattern in membrane potential was changed from one type to another either spontaneously or experimentally. The present results suggest that the variable appearance of the membrane potential trajectory does not represent the different functional category of bulbar post-inspiratory neurons.

Estimating the strength of common input to human motoneurons from the cross-correlogram.

1. The relationship between the motor unit discharge pattern (rate and variability) and synchronization of motor unit pairs was studied in the first dorsal interosseus muscle of human subjects. In separate trials of up to 4 min duration, subjects voluntarily controlled the mean discharge rate of an identified motor unit at one of several prescribed rates (range 7.5-17.5 Hz). 2. The effect of discharge rate on the synchronous peak in the cross-correlogram was examined in eighty motor unit pairs from six subjects. Five commonly used synchronization indices were used to quantify synchrony in the cross-correlograms constructed from different discharge-rate trials. For each synchronization index, the apparent magnitude of synchrony increased at lower motor unit discharge rates. The synchronization indices were not equally sensitive to discharge rate; increases in the different indices ranged from 72 to 494% between the highest and lowest discharge rates. 3. A model of the membrane potential trajectory underlying rhythmic motoneuron discharge was used to explain the observed increase in the magnitude of the synchronization indices at lower discharge rates. The essential feature of this model is that the probability of a common-input EPSP causing a synchronous discharge in two motoneurons is independent of discharge rate. This means that the number of synchronous action potentials in excess of chance in any trial depends on the properties of the common-input EPSPs and the duration of the trial, but is not related to motor unit discharge rates. The model also demonstrated that when the excess synchronous counts are normalized to motor unit discharge rate, or baseline counts in the histogram (as in the conventional synchronization indices), the magnitude of the index increases when the motor unit discharge rates are low. 4. The strength of common input to motoneurons could be misinterpreted if conventional synchronization indices are used because of discharge-rate effects. The model was used to derive an index of the strength of common input to motoneurons (CIS) that was independent of motor unit discharge rate. CIS is the frequency of synchronous action potentials in the motor unit pair in excess of those expected due to chance (calculated during periods of tonic discharge in both units). The mean CIS in first dorsal interosseus motor unit pairs ranged from 0.052 to 1.005 extra synchronous action potentials per second across subjects. 5. Discharge variability was correlated with each of the synchronization indices and the CIS.(ABSTRACT TRUNCATED AT 400 WORDS)

Synaptic interaction between medullary respiratory neurones during apneusis induced by NMDA-receptor blockade in cat.

1. Termination of inspiration is an essential component of respiratory rhythm generation and its perturbation can result in apneusis, i.e. significant prolongation of mechanisms, we studied the postsynaptic events in respiratory neurones during apneustic respiratory periods, and compared them to normal respiratory cycles. 2. Experiments were performed in pentobarbitone-anaesthetized, paralysed, thoracotomized cats ventilated with a constant volume or a cycle-triggered constant pressure pump. Apneusis, separated by normal cycles, was induced as follows: the animal was ventilated by a cycle-triggered pump that normally inflated the lungs during the inspiratory burst of phrenic nerve discharge. The NMDA-receptor blocker MK-801 [(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5, 10-iminemaleate] (0.3-0.7 mg/kg) was administered intravenously, and, for designated breaths, inflation of the lungs was withheld during neural inspiration. 3. Membrane potential trajectories of forty-one late expiratory (E-2) and eight postinspiratory (PI) neurones of the caudal ventral respiratory group were analysed before and/or after MK-801 administration, during normal and apneustic periods. 4. Before MK-801 administration, withholding lung inflation caused modest (10-20%) lengthening of the inspiratory period; after MK-801 administration, withholding inflation caused apneusis. Provided that the lungs were inflated during the inspiratory phase, the temporal pattern of phrenic nerve, recurrent laryngeal nerve and membrane potential trajectories of E-2 and PI neurones were not significantly altered by MK-801. Apneusis following NMDA-receptor blockade produced consistent changes in the synaptic activation patterns of E-2 neurones. In particular, the slow late inspiratory-related depolarization pattern of E-2 neurones was consistently retarded during apneustic inspiratory phases when compared to normal inspiratory phases. This was due to continuation of Cl(-)-mediated synaptic inhibition of E-2 neurones. Superior laryngeal nerve stimulation stopped apneusis and sustained membrane hyperpolarization of E-2 neurones similar to lung inflation. 5. During the plateau phase of apneusis, correlated 10-20 Hz oscillations could be observed in the integrated phrenic and recurrent laryngeal nerve activities as well as in the membrane potential of E-2 neurones. 6. We conclude that: (i) the prolonged inhibition of E-2 neurones during apneusis is indicative of the process responsible for the prolongation of the inspiratory phase.(ABSTRACT TRUNCATED AT 400 WORDS)

Identified proprioceptive afferents and motor rhythm entrainment in the crayfish walking system.

1. In crayfish, Pacifastacus leniusculus, remotion of a walking leg stretches the thoraco-coxal (TC) muscle receptor organ (TCMRO), located at the leg's articulation with the thorax. In vitro, alternate stretch and release of the fourth leg's TCMRO entrained the centrally generated rhythmic motor output to that leg, with the remotor phase of the rhythm entraining to TCMRO stretch, the promoter phase to release. This coordination of motor bursts to afferent input corresponds to that of active, rhythmic movements in vivo. 2. Entrainment was rapid in onset (stable coordination resulting within the first or second stimulus cycle) and was relatively phase-constant (whatever the stimulus frequency, during 1:1 entrainment, remotor bursts began near the onset of stretch and promotor bursts began near the onset of release). Outside the range of 1:1 entrainment, 2:1, 1:2, and 1:3 coordination ratios (rhythm:stimulus) were encountered. Resetting by phasic stimulation of the TCMRO was complete and probabilistic: effective stimuli triggered rapid transitions between the two burst phases. 3. The TCMRO is innervated by two afferents, the nonspiking S and T fibers, which generate graded depolarizing receptor potentials in response to stretch. During proprioceptive entrainment, the more phasic T fiber depolarized and hyperpolarized more rapidly or in advance of the more tonic S fiber. These receptor potentials were modified differently in the two afferents by interaction with central synaptic inputs that were phase-locked to the entrained motor rhythm. 4. Injecting slow sinusoidal current into either afferent alone could entrain motor rhythms: promoter phase bursts were entrained to depolarization of the S fiber or hyperpolarization of the T fiber, whereas the converse response was obtained for remotor phase bursts. 5. During proprioceptive entrainment, tonic hyperpolarization of the S fiber weakened entrained promotor bursts and allowed remotor burst durations to increase. Hyperpolarizing the T fiber weakened entrained remotor bursts and allowed promotor bursts to occur during stretch. These results suggest that the staggered receptor potentials of the two afferents alternately excite opposite burst phases of the rhythm during proprioceptive entrainment. 6. Injecting brief current pulses into either afferent perturbed the timing of entrained bursts in opposite ways, suggesting that, during proprioceptive entrainment, the membrane potential trajectories of the two afferents have reciprocal triggering effects on burst transitions. 7. We infer that entrainment results from 1) complete resetting of burst transitions in a two-phase central oscillator, 2) opposing feedback pathways mediated by a phasic and a tonic afferent, 3) temporally staggered afferent receptor potentials, and 4) the ability of afferent receptor potentials to trigger burst transitions.

Inhibition of caudal medullary expiratory neurones by retrofacial inspiratory neurones in the cat.

1. Comparisons between the spike discharge of inspiratory neurons within the retrofacial area (RFN), and the membrane potential of expiratory neurones within the caudal medulla were made in pentobarbitone-anaesthetized, vagotomized, artificially ventilated cats. Spike-triggered averaging (STA) of synaptic potentials, triggered by the discharge of inspiratory RFN neurones, was utilized to test for synaptic connectivity. 2. Eighty-nine neurons with respiratory-phased discharge patterns were recorded in the vicinity of the RFN. Fifty-four neurones discharged at or slightly before the onset of the inspiratory burst activity of the phrenic nerve and continued firing throughout inspiration. Two continued to fire during post-inspiration. Forty-five of fifty-four inspiratory RFN neurones exhibited incrementing discharge patterns, six discharged with a plateau pattern, while only three neurones had a decrementing discharge pattern. 3. The membrane potential trajectories of caudal expiratory neurones revealed a typical wave of early inspiratory hyperpolarization. Occasionally, a second wave of hyperpolarization occurred during late inspiration, in conjunction with increased phrenic nerve activity. 4. Spike-triggered averaging revealed averaged inhibitory postsynaptic potentials (IPSPs), indicative of inhibitory synaptic connections, between eight and sixty-three pairs of RFN inspiratory and caudal expiratory neurones. 5. Inhibitory postsynaptic potentials detected by STA exhibited a relatively long latency and a slow time course. The IPSPs began, on average, 3.8 ms after an RFN action potential. The rise times, half-widths and durations of IPSPs were longer than expected for a monosynaptic somal input from myelinated axons of inspiratory RFN neurones. It is suggested that an inhibitory relay neurone in the immediate vicinity of the expiratory neurones is activated by a collateral of the RFN inspiratory neurone. 6. Retrofacial inspiratory neurones were antidromically activated only when high-intensity electrical stimulation was applied in the vicinity of caudal expiratory neurones. 7. The averaged IPSPs were preceded by diphasic and triphasic 'spike potentials'. The averaged spike potentials were highly entrained to the action potentials of RFN inspiratory neurones which triggered IPSPs. The spike potentials may be terminal potentials recorded from axons of RFN inspiratory neurones. 8. Evidence for convergence of synaptic inputs was obtained from STA tests in a caudal expiratory neurone receiving IPSPs from four RFN neurones. 9. The functional significance of this observation is discussed. We conclude that RFN inspiratory neurones exert a moderate inhibitory influence and act conjointly with other types of medullary inspiratory neurones.

Synaptic events in ventral respiratory neurones during apnoea induced by laryngeal nerve stimulation in neonatal pig.

1. Postsynaptic potentials evoked by electrical stimulation of superior laryngeal nerve (SLN) were recorded during SLN-induced apnoea from the respiratory neurones of the ventral respiratory group (VRG) in pentobarbitone-anaesthetized, vagotomized and artificially ventilated newborn piglets (n = 14, 4-7 days old). All recorded inspiratory (n = 10), post-inspiratory (n = 10) and expiratory (n = 20) neurones had a triphasic pattern of membrane potential and were identified for their projections to the spinal cord or cervical vagus nerve. 2. During long-lasting apnoea, induced by SLN stimulation, the membrane potential trajectory of each type of recorded neurone was held at the level corresponding approximately to the membrane potential reached during stage I of expiration. Compound postsynaptic potentials evoked in most respiratory-related neurones had an early short-lasting and a late long-lasting component. 3. Postsynaptic potentials in four out of seven inspiratory neurones, in which postsynaptic potentials were well demonstrated, were characterized by an early depolarization followed by long-lasting hyperpolarization. In three other inspiratory neurones only late hyperpolarization was present. The reversal of the late hyperpolarization by intracellular chloride injection was achieved to a different degree in the early and late portions of late hyperpolarization. 4. Postsynaptic potentials evoked in expiratory neurones were studied in sixteen neurones and displayed two patterns: early hyperpolarization followed by long-lasting hyperpolarization (n = 7, six were not antidromically activated after spinal cord stimulation) or early hyperpolarization followed by late depolarization (n = 9, eight projected to the spinal cord). The early hyperpolarization was readily reversed by chloride injection. The late hyperpolarization was more difficult to reverse and usually the reversal was not completed. 5. Postsynaptic potentials evoked in post-inspiratory neurones showed a pattern of two consecutive phases of depolarization. 6. The present study revealed that during long-lasting apnoea evoked by SLN stimulation each category of VRG respiratory neurones received a temporally synchronized combination of an initial fast input derived reflexly from laryngeal afferents, and of late inputs representing involvement of the whole respiratory network in the response.

Patterns of membrane potentials and distributions of the medullary respiratory neurons in the decerebrate rat

We analyzed the membrane potential of 161 respiratory neurons in the medulla of decerebrate rats which were paralyzed and ventilated. Three types of inspiratory (I) neurons were observed: those displaying progressive depolarization in inspiration (augmenting I neurons), those which gradually repolarized after maximal depolarization at the onset of inspiration (decrementing I neurons) and those exhibiting a plateau or bell-shaped membrane potential trajectory throughout inspiration (I-all neurons). Three types of expiratory (E) neurons were also encountered: those in which the membrane potential progressively depolarized (augmenting E neurons), those in which the membrane potential repolarized during the interval between phrenic bursts (decrementing E or post-I neurons) and those exhibiting a plateau or bell-shaped membrane potential trajectory throughout expiration (E-all neurons). Axonal projections of these medullary neurons were identified in the cranial nerves ( n = 34), or in the spinal cord ( n = 19) as revealed by antidromic stimulation and/or by reconstruction following horseradish peroxidase (HRP) labeling. The other 108 neurons were not antidromically activated (NAA) by the stimulations tested, or had their axons terminating inside the medulla as revealed by HRP labeling. All these respiratory neurons, except for 3 which were hypoglossal motoneurons, had their somata within the ventrolateral medulla, in the region of the nucleus ambiguus, homologous to the ventral respiratory group (VRG) of the cat. No dorsal respiratory group (DRG) was detected within the medulla of the rats. Due to this absence of a DRG, it is concluded that the neural organization of respiratory centers is quite different in cats and rats.