Hidehiko Koizumi

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/Cm) in intrinsic oscillatory bursters and intrinsically nonbursters, the two main electrophysiological phenotypes of inspiratory neurons within the pre-BötC. Densities of gNaP were significantly higher for intrinsic bursters, whereas leak conductance densities were not significantly different between intrinsic bursters and nonbursters. By pharmacologically manipulating gNaP and/or gLeak 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 gNaP and gLeak 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 Ca2+-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.

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 ∼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.

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.

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.

Comparison of Surgical and Nonsurgical Treatment of Bilateral Condylar Fractures Based on Maximal Mouth Opening

Abstract 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.

Temporal patterns of trigeminal respiratory activity in rat brainstem-spinal cord in vitro

Respiratory activity in trigeminal (V) motoneurons was studied in rhythmically active en bloc brainstem-spinal cord preparations isolated from neonatal rats (P0-P3). In the majority of preparations (83%), the temporal pattern of V activity consisted of spontaneous inspiratory phasic discharge with onset delayed or coincident with onset of phrenic motoneuron discharge. Blockade of alpha-2 noradrenergic receptor activation shifted onset of V respiratory discharges earlier than phrenic discharges, while elevation of extracellular potassium concentration or blockade of GABAergic and glycinergic inhibitory synaptic transmission had little effect on temporary pattern of V respiratory discharges. We conclude V motoneurons in the in vitro preparation generate respiratory activity during inspiratory phase, and their temporal patterns are modulated by inhibitory noradrenergic synaptic transmission.

Respiratory Activities in Relation to Palatal Muscle Contraction

This study investigated the activities of the tensor and levator veli palatini muscles related to respiration. During quiet breathing, no activity was observed in either muscle. With either hypercapnic or hypoxemic condition, the tensor veli palatini muscle exhibited phasic activity during inspiration. The levator veli palatini muscle showed phasic activity during expiration with hypoxemia (PaO2 less than 40 mm Hg). NaCN perfused bilaterally through the carotid sinus induced these respiratory activities. The tensor veli palatini muscle was more sensitive than the levator veli palatini muscle to NaCN.

Evidence of aerosolised floating blood mist during oral surgery

Dental surgery performed with high speed instruments, such as a dental turbine, air motor, or micro-engine handpiece, produces a large amount of splattering and particles, which can be contaminated by micro-organisms from the oral cavity. It has been speculated that such particulate mists contain blood-based elements. In the present study, we investigated whether blood-contaminated aerosol was present in a room where oral surgery was performed with high speed instruments. An extra-oral evacuator system was used for sample collection (N=132). For the experiment, a non-woven towel was set on the nozzle of the evacuator as a filter and invisible mist was collected at distances of 20, 60 and 100 cm from the surgical site. A leucomalachite green presumptive test was performed with each filter after every tooth extraction. At locations 20 and 100 cm from the surgical site, 76% and 57%, respectively, of the particulates were positive in blood presumptive tests. Based on our results, we consider that blood-contaminated materials have the potential to be suspended in air as blood-contaminated aerosol. These results indicate the risk of cross-infection at the dental practice for immunocompromised patients as well as healthy personnel.