Organization of the core respiratory network: Insights from optogenetic and modeling studies.

The circuit organization within the mammalian brainstem respiratory network, specifically within and between the pre-Bötzinger (pre-BötC) and Bötzinger (BötC) complexes, and the roles of these circuits in respiratory pattern generation are continuously debated. We address these issues with a combination of optogenetic experiments and modeling studies. We used transgenic mice expressing channelrhodopsin-2 under the VGAT-promoter to investigate perturbations of respiratory circuit activity by site-specific photostimulation of inhibitory neurons within the pre-BötC or BötC. The stimulation effects were dependent on the intensity and phase of the photostimulation. Specifically: (1) Low intensity (≤ 1.0 mW) pulses delivered to the pre-BötC during inspiration did not terminate activity, whereas stronger stimulations (≥ 2.0 mW) terminated inspiration. (2) When the pre-BötC stimulation ended in or was applied during expiration, rebound activation of inspiration occurred after a fixed latency. (3) Relatively weak sustained stimulation (20 Hz, 0.5-2.0 mW) of pre-BötC inhibitory neurons increased respiratory frequency, while a further increase of stimulus intensity (> 3.0 mW) reduced frequency and finally (≥ 5.0 mW) terminated respiratory oscillations. (4) Single pulses (0.2-5.0 s) applied to the BötC inhibited rhythmic activity for the duration of the stimulation. (5) Sustained stimulation (20 Hz, 0.5-3.0 mW) of the BötC reduced respiratory frequency and finally led to apnea. We have revised our computational model of pre-BötC and BötC microcircuits by incorporating an additional population of post-inspiratory inhibitory neurons in the pre-BötC that interacts with other neurons in the network. This model was able to reproduce the above experimental findings as well as previously published results of optogenetic activation of pre-BötC or BötC neurons obtained by other laboratories. The proposed organization of pre-BötC and BötC circuits leads to testable predictions about their specific roles in respiratory pattern generation and provides important insights into key circuit interactions operating within brainstem respiratory networks.

Structural-functional properties of identified excitatory and inhibitory interneurons within pre-Botzinger complex respiratory microcircuits.

We comparatively analyzed cellular and circuit properties of identified rhythmic excitatory and inhibitory interneurons within respiratory microcircuits of the neonatal rodent pre-Bötzinger complex (pre-BötC), the structure generating inspiratory rhythm in the brainstem. We combined high-resolution structural-functional imaging, molecular assays for neurotransmitter phenotype identification in conjunction with electrophysiological property phenotyping, and morphological reconstruction of interneurons in neonatal rat and mouse slices in vitro. This approach revealed previously undifferentiated structural-functional features that distinguish excitatory and inhibitory interneuronal populations. We identified distinct subpopulations of pre-BötC glutamatergic, glycinergic, GABAergic, and glycine-GABA coexpressing interneurons. Most commissural pre-BötC inspiratory interneurons were glutamatergic, with a substantial subset exhibiting intrinsic oscillatory bursting properties. Commissural excitatory interneurons projected with nearly planar trajectories to the contralateral pre-BötC, many also with axon collaterals to areas containing inspiratory hypoglossal (XII) premotoneurons and motoneurons. Inhibitory neurons as characterized in the present study did not exhibit intrinsic oscillatory bursting properties, but were electrophysiologically distinguished by more pronounced spike frequency adaptation properties. Axons of many inhibitory neurons projected ipsilaterally also to regions containing inspiratory XII premotoneurons and motoneurons, whereas a minority of inhibitory neurons had commissural axonal projections. Dendrites of both excitatory and inhibitory interneurons were arborized asymmetrically, primarily in the coronal plane. The dendritic fields of inhibitory neurons were more spatially compact than those of excitatory interneurons. Our results are consistent with the concepts of a compartmental circuit organization, a bilaterally coupled excitatory rhythmogenic kernel, and a role of pre-BötC inhibitory neurons in shaping inspiratory pattern as well as coordinating inspiratory and expiratory activity.

Predictions and experimental tests of a new biophysical model of the mammalian respiratory oscillator.

Previously our computational modeling studies (Phillips et al., 2019) proposed that neuronal persistent sodium current (INaP) and calcium-activated non-selective cation current (ICAN) are key biophysical factors that, respectively, generate inspiratory rhythm and burst pattern in the mammalian preBötzinger complex (preBötC) respiratory oscillator isolated in vitro. Here, we experimentally tested and confirmed three predictions of the model from new simulations concerning the roles of INaP and ICAN: (1) INaP and ICAN blockade have opposite effects on the relationship between network excitability and preBötC rhythmic activity; (2) INaP is essential for preBötC rhythmogenesis; and (3) ICAN is essential for generating the amplitude of rhythmic output but not rhythm generation. These predictions were confirmed via optogenetic manipulations of preBötC network excitability during graded INaP or ICAN blockade by pharmacological manipulations in slices in vitro containing the rhythmically active preBötC from the medulla oblongata of neonatal mice. Our results support and advance the hypothesis that INaP and ICAN mechanistically underlie rhythm and inspiratory burst pattern generation, respectively, in the isolated preBötC.

TASK channels contribute to the K+-dominated leak current regulating respiratory rhythm generation in vitro.

Leak channels regulate neuronal activity and excitability. Determining which leak channels exist in neurons and how they control electrophysiological behavior is fundamental. Here we investigated TASK channels, members of the two-pore domain K(+) channel family, as a component of the K(+)-dominated leak conductance that controls and modulates rhythm generation at cellular and network levels in the mammalian pre-Bötzinger complex (pre-BötC), an excitatory network of neurons in the medulla critically involved in respiratory rhythmogenesis. By voltage-clamp analyses of pre-BötC neuronal current-voltage (I-V) relations in neonatal rat medullary slices in vitro, we demonstrated that pre-BötC inspiratory neurons have a weakly outward-rectifying total leak conductance with reversal potential that was depolarized by approximately 4 mV from the K(+) equilibrium potential, indicating that background K(+) channels are dominant contributors to leak. This K(+) channel component had I-V relations described by constant field theory, and the conductance was reduced by acid and was augmented by the volatile anesthetic halothane, which are all hallmarks of TASK. We established by single-cell RT-PCR that pre-BötC inspiratory neurons express TASK-1 and in some cases also TASK-3 mRNA. Furthermore, acid depolarized and augmented bursting frequency of pre-BötC inspiratory neurons with intrinsic bursting properties. Microinfusion of acidified solutions into the rhythmically active pre-BötC network increased network bursting frequency, halothane decreased bursting frequency, and acid reversed the depressant effects of halothane, consistent with modulation of network activity by TASK channels. We conclude that TASK-like channels play a major functional role in chemosensory modulation of respiratory rhythm generation in the pre-Bötzinger complex in vitro.

Respiratory rhythm generation during gasping depends on persistent sodium current.

In severe hypoxia, homeostatic mechanisms maintain function of the brainstem respiratory network. We hypothesized that hypoxia involves a transition from neuronal mechanisms of normal breathing (eupnea) to a rudimentary pattern of inspiratory movements (gasping). We provide evidence for hypoxia-driven transformation within the central respiratory oscillator, in which gasping relies on persistent sodium current, whereas eupnea does not depend on this cellular mechanism.

Persistent Na+ and K+-dominated leak currents contribute to respiratory rhythm generation in the pre-Bötzinger complex in vitro.

A central problem in analyzing neural circuit function is establishing how intrinsic neuronal conductances contribute to the generation of network activity. We used real-time calcium activity imaging combined with whole-cell patch-clamp recording to analyze contributions of subthreshold conductances in the excitatory rhythm-generating network in the respiratory pre-Bötzinger complex (pre-BötC) of neonatal rat in vitro brainstem slice preparations. Voltage-clamp ramp recordings from imaged pre-BötC neurons revealed that persistent sodium (NaP) and K+-dominated leak currents primarily contribute to subthreshold I-V relations. We quantified NaP and leak conductance densities (g/C(m)) in intrinsic oscillatory bursters and intrinsically nonbursters, the two main electrophysiological phenotypes of inspiratory neurons within the pre-BötC. Densities of g(NaP) were significantly higher for intrinsic bursters, whereas leak conductance densities were not significantly different between intrinsic bursters and nonbursters. By pharmacologically manipulating g(NaP) and/or g(Leak) directly within the pre-BötC, we could modulate network oscillation frequency over a wide dynamic range and cause transitions between oscillatory and quiescent states. These results were consistent with models of the pre-BötC excitatory network consisting of heterogeneous mixtures of intrinsic bursters and nonintrinsic bursters incorporating g(NaP) and g(Leak) with parameter values found experimentally. We propose a paradigm whereby NaP and Leak represent a functional set of subthreshold conductances that endow the pre-BötC with rhythmogenic properties and represent targets for modulatory control of inspiratory rhythm generation.

Functional imaging, spatial reconstruction, and biophysical analysis of a respiratory motor circuit isolated in vitro.

We combined real-time calcium-based neural activity imaging with whole-cell patch-clamp recording techniques to map the spatial organization and analyze electrophysiological properties of respiratory neurons forming the circuit transmitting rhythmic drive from the pre-Bötzinger complex (pre-BötC) through premotoneurons to hypoglossal (XII) motoneurons. Inspiratory pre-BötC neurons, XII premotoneurons (preMNs), and XII motoneurons (MNs) were retrogradely labeled with Ca(2+)-sensitive dye in neonatal rat in vitro brainstem slices. PreMN cell bodies were arrayed dorsomedially to pre-BötC neurons with little spatial overlap; axonal projections to MNs were ipsilateral. Inspiratory MNs were distributed in dorsal and ventral subnuclei of XII. Voltage-clamp recordings revealed that two currents, persistent sodium current (NaP) and K(+)-dominated leak current (Leak), primarily contribute to preMN/MN subthreshold current-voltage relationships. NaP or Leak conductance densities in preMNs and MNs were not significantly different. We quantified preMN and MN action potential time course and spike frequency-current (f-I) relationships and found no significant differences in repetitive spiking dynamics, steady-state f-I gains, and afterpolarizing potentials. Rhythmic synaptic drive current densities were similar in preMNs and MNs. Our results indicate that, despite topographic and morphological differences, preMNs and MNs have some common intrinsic membrane, synaptic integration, and spiking properties that we postulate ensure fidelity of inspiratory drive transmission and conversion of synaptic drive into (pre)motor output. There also appears to be a common architectonic organization for some respiratory drive transmission circuits whereby many preMNs are spatially segregated from pre-BötC rhythm-generating neurons, which we hypothesize may facilitate downstream integration of convergent inputs for premotor pattern formation.

Biophysical mechanisms in the mammalian respiratory oscillator re-examined with a new data-driven computational model.

An autorhythmic population of excitatory neurons in the brainstem pre-Bötzinger complex is a critical component of the mammalian respiratory oscillator. Two intrinsic neuronal biophysical mechanisms-a persistent sodium current ([Formula: see text]) and a calcium-activated non-selective cationic current ([Formula: see text])-were proposed to individually or in combination generate cellular- and circuit-level oscillations, but their roles are debated without resolution. We re-examined these roles in a model of a synaptically connected population of excitatory neurons with [Formula: see text] and [Formula: see text]. This model robustly reproduces experimental data showing that rhythm generation can be independent of [Formula: see text] activation, which determines population activity amplitude. This occurs when [Formula: see text] is primarily activated by neuronal calcium fluxes driven by synaptic mechanisms. Rhythm depends critically on [Formula: see text] in a subpopulation forming the rhythmogenic kernel. The model explains how the rhythm and amplitude of respiratory oscillations involve distinct biophysical mechanisms.

Possible involvement of neurons in locus coeruleus in inhibitory effect on glossopharyngeal expiratory activity in a neonatal rat brainstem-spinal cord preparation in vitro.

In this study, we found that a certain motor branch of glossopharyngeal (IX) motor nerves stably exhibits not only inspiratory activity but also expiratory activity with pons removal in neonatal rat brainstem-spinal cord preparations in vitro. Because this finding indicates that IX expiratory activity is masked by an inhibitory mechanism operating in the pons, we sought to determine the candidate neurons that exert an inhibitory effect on IX expiratory activity. IX expiratory activity was observed when only the pons was perfused with noradrenaline (NA) or clonidine (alpha2 adrenergic receptor agonist), but not when NA and yohimbine (alpha2 adrenergic receptor antagonist) were perfused together. IX expiratory activity was also observed following the removal of the dorsal pons but not the ventral pons. The local administration of clonidine into the bilateral locus coeruleus (LC) evoked burst discharges during the expiratory phase in the IX motor rootlet. These results suggest that neurons in the LC that possess an alpha2 adrenergic receptor on the membrane surface exert a tonic inhibitory effect on IX expiratory activity in neonatal rat brainstem-spinal cord preparations.

High incidence of blood exposure due to imperceptible contaminated splatters during oral surgery.

PURPOSE: To evaluate the incidence of blood exposure during outpatient oral surgery from splattering caused by use of high-speed rotary instruments at the Referral and Teaching Center, University Dental Hospital. MATERIALS AND METHODS: Twenty-five consecutive patients who had impacted mandibular third molars were selected. The attending surgeon wore an operation gown and visor mask, and carried out the tooth extraction with the regular procedure. We counted the number of bloodstains found on the operation gown and visor mask, and confirmed the presence of diluted and invisible bloodstains using a leucomalachite green presumptive test, which was able to detect dilutions up to 1:4,000. RESULTS: There were 469 separate bloodstains on the gown and visor mask of oral surgeons, which came from 19 (76%) of 25 patients during impacted mandibular third molar surgery. Presumptive tests for invisible bloodstains resulted in 1,206 positive reactions, 2.57-fold greater than the visible stains, from 88% of the cases. All of the surgeons were right-handed and the common areas of staining were the right forearm, face, and thorax regions. CONCLUSIONS: Dental procedures with high-speed instruments exposed surgeons to possible blood-borne infections by splashing in nearly 90% of the cases. Greater than 50% of the stains were invisible to the naked eye. Based on our results, strict compliance with barrier precautions, including routine use of an operation gown and visor mask, is recommended whenever oral surgery is carried out with high-speed rotary instruments.

Kinetic properties of persistent Na+ current orchestrate oscillatory bursting in respiratory neurons.

The rhythmic pattern of breathing depends on the pre-Bötzinger complex (preBötC) in the brainstem, a vital circuit that contains a population of neurons with intrinsic oscillatory bursting behavior. Here, we investigate the specific kinetic properties that enable voltage-gated sodium channels to establish oscillatory bursting in preBötC inspiratory neurons, which exhibit an unusually large persistent Na+ current (INaP). We first characterize the kinetics of INaP in neonatal rat brainstem slices in vitro, using whole-cell patch-clamp and computational modeling, and then test the contribution of INaP to rhythmic bursting in live neurons, using the dynamic clamp technique. We provide evidence that subthreshold activation, persistence at suprathreshold potentials, slow inactivation, and slow recovery from inactivation are kinetic features of INaP that regulate all aspects of intrinsic rhythmic bursting in preBötC neurons. The slow and cumulative inactivation of INaP during the burst active phase controls burst duration and termination, while the slow recovery from inactivation controls the duration of the interburst interval. To demonstrate this mechanism, we develop a Markov state model of INaP that explains a comprehensive set of voltage clamp data. By adding or subtracting a computer-generated INaP from a live neuron via dynamic clamp, we are able to convert nonbursters into intrinsic bursters, and vice versa. As a control, we test a model with inactivation features removed. Adding noninactivating INaP into nonbursters results in a pattern of random transitions between sustained firing and quiescence. The relative amplitude of INaP is the key factor that separates intrinsic bursters from nonbursters and can change the fraction of intrinsic bursters in the preBötC. INaP could thus be an important target for regulating network rhythmogenic properties.

Transient Receptor Potential Channels TRPM4 and TRPC3 Critically Contribute to Respiratory Motor Pattern Formation but not Rhythmogenesis in Rodent Brainstem Circuits.

Transient receptor potential channel, TRPM4, the putative molecular substrate for Ca2+-activated nonselective cation current (ICAN), is hypothesized to generate bursting activity of pre-Bötzinger complex (pre-BötC) inspiratory neurons and critically contribute to respiratory rhythmogenesis. Another TRP channel, TRPC3, which mediates Na+/Ca2+ fluxes, may be involved in regulating Ca2+-related signaling, including affecting TRPM4/ICAN in respiratory pre-BötC neurons. However, TRPM4 and TRPC3 expression in pre-BötC inspiratory neurons and functional roles of these channels remain to be determined. By single-cell multiplex RT-PCR, we show mRNA expression for these channels in pre-BötC inspiratory neurons in rhythmically active medullary in vitro slices from neonatal rats and mice. Functional contributions were analyzed with pharmacological inhibitors of TRPM4 or TRPC3 in vitro as well as in mature rodent arterially perfused in situ brainstem-spinal cord preparations. Perturbations of respiratory circuit activity were also compared with those by a blocker of ICAN. Pharmacologically attenuating endogenous activation of TRPM4, TRPC3, or ICANin vitro similarly reduced the amplitude of inspiratory motoneuronal activity without significant perturbations of inspiratory frequency or variability of the rhythm. Amplitude perturbations were correlated with reduced inspiratory glutamatergic pre-BötC neuronal activity, monitored by multicellular dynamic calcium imaging in vitro. In more intact circuits in situ, the reduction of pre-BötC and motoneuronal inspiratory activity amplitude was accompanied by reduced post-inspiratory motoneuronal activity, without disruption of rhythm generation. We conclude that endogenously activated TRPM4, which likely mediates ICAN, and TRPC3 channels in pre-BötC inspiratory neurons play fundamental roles in respiratory pattern formation but are not critically involved in respiratory rhythm generation.

Comparison of surgical and nonsurgical treatment of bilateral condylar fractures based on maximal mouth opening.

This study presents a comparative analysis of the open surgical and nonsurgical treatment of patients with bilateral condylar fractures. Sixty-seven (67) patients were treated, and the completed data on 55 patients were reviewed to compare both therapeutic modalities, which consisted of nonsurgical and surgical treatment in 37 and 18 patients, respectively. In the nonsurgical group, 23 patients (23/37, 62%) had normal mouth opening. Functional success rate was 79% (15/19) and 44% (8/18) in young adult patients (-29yrs) and older patients (30+yrs), respectively, and there was a significant difference of outcome between the two groups. In nonsurgically treated young patients with disorders, bilateral dislocation and existence of concomitant mandibular fractures were commonly observed. In the open surgical group, seven (7/11, 64%) young adult and three (3/7, 43%) older patients gained normal mouth opening, and no significant difference was observed. Additionally, there was no difference between non-surgical and surgical treatment in any category. Patients undergoing rigid fixation benefited from restoring maximum mouth opening, although there was no significant difference between the rigid and non-rigid fixation groups. Based on these findings, nonrigid fixation should be avoided, and rigid fixation might improve outcome in young adult patients with severe fracture pattern, such as bilateral dislocation and concomitant mandibular fracture.

The respiratory control mechanisms in the brainstem and spinal cord: integrative views of the neuroanatomy and neurophysiology.

Respiratory activities are produced by medullary respiratory rhythm generators and are modulated from various sites in the lower brainstem, and which are then output as motor activities through premotor efferent networks in the brainstem and spinal cord. Over the past few decades, new knowledge has been accumulated on the anatomical and physiological mechanisms underlying the generation and regulation of respiratory rhythm. In this review, we focus on the recent findings and attempt to elucidate the anatomical and functional mechanisms underlying respiratory control in the lower brainstem and spinal cord.

Voltage-Dependent Rhythmogenic Property of Respiratory Pre-Bötzinger Complex Glutamatergic, Dbx1-Derived, and Somatostatin-Expressing Neuron Populations Revealed by Graded Optogenetic Inhibition.

The rhythm of breathing in mammals, originating within the brainstem pre-Bötzinger complex (pre-BötC), is presumed to be generated by glutamatergic neurons, but this has not been directly demonstrated. Additionally, developmental expression of the transcription factor Dbx1 or expression of the neuropeptide somatostatin (Sst), has been proposed as a marker for the rhythmogenic pre-BötC glutamatergic neurons, but it is unknown whether these other two phenotypically defined neuronal populations are functionally equivalent to glutamatergic neurons with regard to rhythm generation. To address these problems, we comparatively investigated, by optogenetic approaches, the roles of pre-BötC glutamatergic, Dbx1-derived, and Sst-expressing neurons in respiratory rhythm generation in neonatal transgenic mouse medullary slices in vitro and also more intact adult perfused brainstem-spinal cord preparations in situ. We established three different triple-transgenic mouse lines with Cre-driven Archaerhodopsin-3 (Arch) expression selectively in glutamatergic, Dbx1-derived, or Sst-expressing neurons for targeted photoinhibition. In each line, we identified subpopulations of rhythmically active, Arch-expressing pre-BötC inspiratory neurons by whole-cell recordings in medullary slice preparations in vitro, and established that Arch-mediated hyperpolarization of these inspiratory neurons was laser power dependent with equal efficacy. By site- and population-specific graded photoinhibition, we then demonstrated that inspiratory frequency was reduced by each population with the same neuronal voltage-dependent frequency control mechanism in each state of the respiratory network examined. We infer that enough of the rhythmogenic pre-BötC glutamatergic neurons also have the Dbx1 and Sst expression phenotypes, and thus all three phenotypes share the same voltage-dependent frequency control property.

Perturbations of Respiratory Rhythm and Pattern by Disrupting Synaptic Inhibition within Pre-Bötzinger and Bötzinger Complexes.

The pre-Bötzinger (pre-BötC) and Bötzinger (BötC) complexes are the brainstem compartments containing interneurons considered to be critically involved in generating respiratory rhythm and motor pattern in mammals. Current models postulate that both generation of the rhythm and coordination of the inspiratory-expiratory pattern involve inhibitory synaptic interactions within and between these regions. Both regions contain glycinergic and GABAergic neurons, and rhythmically active neurons in these regions receive appropriately coordinated phasic inhibition necessary for generation of the normal three-phase respiratory pattern. However, recent experiments attempting to disrupt glycinergic and GABAergic postsynaptic inhibition in the pre-BötC and BötC in adult rats in vivo have questioned the critical role of synaptic inhibition in these regions, as well as the importance of the BötC, which contradicts previous physiological and pharmacological studies. To further evaluate the roles of synaptic inhibition and the BötC, we bilaterally microinjected the GABAA receptor antagonist gabazine and glycinergic receptor antagonist strychnine into the pre-BötC or BötC in anesthetized adult rats in vivo and in perfused in situ brainstem-spinal cord preparations from juvenile rats. Muscimol was microinjected to suppress neuronal activity in the pre-BötC or BötC. In both preparations, disrupting inhibition within pre-BötC or BötC caused major site-specific perturbations of the rhythm and disrupted the three-phase motor pattern, in some experiments terminating rhythmic motor output. Suppressing BötC activity also potently disturbed the rhythm and motor pattern. We conclude that inhibitory circuit interactions within and between the pre-BötC and BötC critically regulate rhythmogenesis and are required for normal respiratory motor pattern generation.

Localization of premotoneurons for an NMDA-induced repetitive rhythmical activity to TMNs.

The localization or characteristics of the premotoneurons for trigeminal rhythmical activity have not been clarified. We investigated the localization of premotoneurons generating an NMDA-induced repetitive rhythmical activity to trigeminal motoneurons (TMNs). The minimal circuitry for this rhythmical activity was determined using a fragmented slice preparation of the isolated brain stem from neonatal rats (0-3 days old). We recorded rhythmical neural activities from TMNs using whole and fragmented brainstem slices preparation including the trigeminal motor nucleus in the presence of the excitatory amino acid agonist NMA and the GABAA receptor antagonist, bicuculline methiodide (BIC). TMNs receive projections from premotoneurons for an NMDA-induced rhythmical activity, which can be located in the area 300 microm surrounding the trigeminal motor nucleus. NMA (20 microM) and BIC (10 microM) induced repetitive rhythmical activities on TMNs.

Swallowing-like activity elicited in vitro in neonatal rat organ attached brainstem block preparation.

The purpose of this study was to induce swallowing in an in vitro neonatal rat brainstem preparation and to analyze the circuit. When we applied GABA(A) receptor antagonist (bicuculine methiodide, BIC) into the the nucleus tractus solitarius (NTS) in the organ attached brainstem preparation of neonatal (0-3 days after birth) rats, jaw closing movement, palatal lifting, and tongue peristalsis-like movement were seen, subsequent to elevation of the tip of the tongue and anterior movement of the larynx (closure of the trachea). The NTS has been proposed to be a critical locus for swallowing pattern generation in mammals. Electrical stimulation into the NTS or the vagal afferent nerve (X) following an application of BIC (10 microM) to the recording chamber initiated the same organ movement. This movement caused temporary inhibition of respiratory activity that was simultaneously recorded from the fourth cervical ventral nerve (C4). We were also able to elicit this activity in a whole organ (from lip to stomach, midline intact) preparation, whose oral cavity was filled with dye (pontamine sky-blue 3 mM, 50 microl), using each of the three types of stimulation. The esophagus, which was never stained by spontaneous respiratory movements, was stained only after the experimental stimulation. We concluded that the activity elicited was swallowing-like activity and the smallest circuit for swallowing pattern generation exists in this preparation.

Oral-motor patterns of rhythmic trigeminal activity generated in fetal rat brainstem in vitro.

Development of neural circuits generating fetal oral-motor activity was characterized in an in vitro isolated brainstem block preparation. Rhythmical trigeminal activity (RTA) at E20-E21 resembled either the pattern or rhythm of neonatal RTA. Conversely, at E18-E19, RTA displayed a different pattern of discharge from neonatal RTA, and output was not regular but intermittent with another slow rhythm.

Differential discharge patterns of rhythmical activity in trigeminal motoneurons during fictive mastication and respiration in vitro.

Rhythmical activity in trigeminal motoneurons (TMNs) was studied in an in vitro neonatal rat brainstem preparation that retains functionally active circuits for oral-motor behaviors. Whole-cell current-clamp recording from TMNs demonstrated rhythmical activities during both spontaneously generated respiratory activity and neurochemically induced rhythmical oral-motor activity. TMNs showed spontaneous rhythmical (0.08 +/- 0.04 Hz) activities of burst-firing pattern during inspiration synchronized with inspiratory activities recorded in hypoglossal nerves. During rhythmical oral-motor activity induced by bath application of N-methyl-d,l-aspartic acid and the GABA(A) receptor antagonist bicuculline methiodide, TMNs showed only a rhythmical (5.6 +/- 0.8 Hz) pattern of single-spike discharge. TMNs never showed a burst-firing pattern during rhythmical oral-motor activity even when membrane potentials were shifted either to depolarized or hyperpolarized levels. Rhythmical activity in TMNs exhibited different discharge patterns between rhythmical oral-motor activity and respiratory activity generated in vitro.