Effects of hypocapnia on spontaneous burst activity in the piriform-amygdala complex of newborn rat brain preparations in vitro.

We examined effects of hypocapnia on burst activity in the piriform-amygdala complex and C(4) inspiratory activity in limbic-brainstem-spinal cord preparations from 0- to 1-day-old rats. Hypocapnia (2% CO(2)) increased the burst rate in the piriform-amygdala complex but decreased the C(4) inspiratory burst rate. Since hyperventilation induces hypocapnia, and enhanced amygdala activity may be involved in induction of a sense of anxiety, our findings might explain the neuronal mechanism of a vicious circle between hyperventilation and an increased sense of anxiety.

Spontaneous oscillatory burst activity in the piriform-amygdala region and its relation to in vitro respiratory activity in newborn rats.

The amygdala is important for the formation of emotions that are affected by olfactory information. The piriform cortex is involved in information processing related to olfaction. To investigate functional interactions between the piriform cortex and amygdala and their relation to medullary respiratory activity, we developed a novel in vitro preparation including the limbic system, brainstem, and spinal cord of newborn rats. With the use of optical and electrophysiologic recordings, we analyzed spontaneous neuronal activity in the piriform-amygdala complex in limbic-brainstem-spinal cord preparations from 0- to 1-day-old rats. For optical recordings, the preparation was stained with a voltage-sensitive dye, and inspiratory activity was monitored from the fourth cervical (C4) ventral root. Spontaneous oscillatory burst activity (up to 10/min) was detected from the rostral cut surface of limbic and para-limbic regions including the piriform cortex and amygdala. The burst activity initially appeared in the piriform cortex and then propagated to the amygdala. We averaged the imaging data in the limbic area with the use of C4 inspiratory activity as a trigger signal. The results suggest functional coupling of the rhythmic burst activity in the piriform-amygdala complex to medullary inspiratory activity, which was confirmed electrophysiologically by cross-correlation analysis of these signals. This rhythmic burst activity may be involved in the development of neuronal circuits that process information related to olfaction, emotion, and respiration.

Transient O2-dependent effects of CO2 on ventilation in the anesthetized mouse.

In this study we sought to determine the effects of background hyperoxia on the ventilatory response to hypercapnia. We addressed this issue by examining the temporal profile of the first minute transients of minute ventilation, and its frequency and tidal components, in response to 5% and 10% CO2 each co-applied with the natural (balanced with air) and hyperoxic (balanced with O2) levels of oxygen. The study was performed on the urethane-anesthetized, tracheostomized, spontaneously breathing mouse, placed in a flow-through body plethysmograph. We identified an early suppressant effect of CO2-in-O2 on breathing frequency. The frequency declined to 88.5 +/-1.4% and 87.8 +/-1.9% relative to the pre-test, baseline level for 5% and 10% CO2, respectively. There was a compensatory rise in tidal volume and no major change in the overall ventilation. In contrast, CO2-in-Air resulted in ventilatory stimulation caused in equal measure by frequency and tidal components. Thus, the inhibitory effect on breathing frequency of the CO2-in-O2 resulted from the O2 content in the mixture and had the temporal characteristics consistent with carotid body function. In conclusion, transient O2-dependent effects can bear on the nascent hypercapnic ventilatory response. The complexity of the O2-CO2 interaction regarding the breathing pattern components should be taken into account while designing the optimal conditions for a hypercapnic test.

Developmental changes in the spatio-temporal pattern of respiratory neuron activity in the medulla of late fetal rat.

We investigated how the spatio-temporal pattern of respiratory neuron network activity in the ventral medulla changes during the late fetal period of rat. Brainstem-spinal cord preparations isolated from rat fetuses on embryonic days 17-21 (E17-E21) were stained with a voltage-sensitive dye for optical image analysis of neuronal activity of the ventral medulla. The spatio-temporal pattern of respiratory neuron activity in the preparation from E20 to E21 was basically identical to that of neonatal rat; pre-inspiratory activity in a limited region of the rostral ventrolateral medulla, the para-facial region, preceded by several hundred milliseconds the onset of inspiratory activity in the more caudal ventrolateral medulla, the pre-Bötzinger complex level. In contrast, in E17-E18 specimens, pre-inspiratory activity could not be detected in the rostral medulla at the level of the facial nucleus. Neuronal activity appeared to begin at the pre-Bötzinger complex level shortly before onset of the inspiratory burst. Strong activity then developed in the facial nucleus and peaked in the post-inspiratory phase. The transition of these patterns of respiratory activity occurred at E19. We conclude that the changes in the spatio-temporal pattern of neuronal activity reflect developmental changes in the cellular elements underlying rhythm generation in the fetal respiratory neuron network. We suggest that the pre-inspiratory neuron network of the para-facial region in the rostral ventrolateral medulla functions as the rhythm generator after E19/20.

The amygdala of patients with Parkinson's disease is silent in response to fearful facial expressions.

We previously found that patients with Parkinson's disease (PD) were impaired with respect to recognition of fear and disgust in facial expressions. To investigate the neural mechanisms that underlie this impairment, we recorded visual event-related potentials (ERPs) in response to the viewing of fearful facial expressions. Ten normal elderly volunteers and nine patients with PD were studied. Fearful, surprised, and neutral facial expressions were presented randomly for 500 ms each, with a probability of 0.1, 0.1, and 0.8, respectively. The locations of the components of the ERPs were analyzed using a scalp-skull-brain/dipole tracing method. The ERPs elicited in response to the facial stimuli consisted of a negative peak (N1), two positive peaks, and a subsequent slow negative shift. For N1, the equivalent current dipoles were concentrated in the fusiform gyrus, right superior temporal gyrus, parahippocampal gyrus, cingulate cortex, and cerebellum, in normal subjects. In response to the fearful stimulus, dipoles were also generated from the amygdala in seven out of 10 normal subjects. In contrast, in patients with PD, N1 was centered bilaterally in the angular gyrus and supramarginal gyrus, and there was no neuronal activity in the amygdala. After N1, dipoles moved toward the frontal region in normal subjects, whereas they remained in the parietal lobes in patients with PD. These results suggest that neither the amygdala nor the temporal visual-associated cortices are involved in responding to fearful expressions in patients with PD. Corticostriatal connections may be variably affected by a lack of dopamine or by pathological changes in the amygdala. Thus, somatosensory recruitment may overcome the mild cognitive emotional deficits that are present in patients with PD owing to a dysfunction of the amygdala.

Inhibitory effect of pain-eliciting transcutaneous electrical stimulation on vibration-induced finger flexion reflex in the human upper limb.

We studied the effects of non-pain transcutaneous electrical stimulation (TES) and pain-eliciting TES on vibration-induced finger flexion reflex (VFR) in 12 healthy volunteers. Tonic finger flexion reflex in the upper limb was induced by the application of vibratory stimulation on the volar side of the middle fingertip in the right hand before and after TES. Non-pain TES or pain-eliciting TES was applied on the skin between the bases of the first and second metacarpals in the right hand dorsal area in a crossover design. Pain-eliciting TES inhibited VFRs significantly (Fisher's PLSD, p <0.01), compared to those of the time-control group during and after TES. VFRs were reduced approximately to 63.8% and 78.6% of prestimulation flexion force during and after pain-eliciting TES, respectively. Nonpain TES did not inhibit VFR. These results suggest that pain-conducting afferent fibers have inhibitory neuronal connection over the ipsilateral reflex circuits of VFR in the upper limb.

Neuronal histamine release elicited by hyperthermia mediates tracheal dilation and pressor response.

Whether brain histaminergic neurons contribute to the regulation of tracheal tone and peripheral vascular tone under hyperthermia was investigated in anesthetized rabbits. Histamine release from the rostral ventrolateral medulla (RVLM), the raphe nuclei, and the solitary nucleus of the medulla oblongata was significantly increased by hyperthermia. The increased histamine was significantly suppressed by 10(-6) M tetrodotoxin microdialyzed in each area. Tracheal pressure and mean arterial pressure were significantly decreased and increased by hyperthermia, respectively. An H(1)-receptor antagonist, 5 x 10(-6) M (+)-chlorpheniramine, bilaterally microdialyzed in the RVLM significantly enhanced histamine release in the RVLM as well as significantly suppressed tracheal dilation and pressor response caused by hyperthermia. These data indicate that histamine release in the medulla oblongata is enhanced by hyperthermia. The enhanced histamine is the neuronal origin and the cause of tracheal dilation and pressor response at least via H(1) receptors in the RVLM. Brain histaminergic neurons play important roles in tracheal tone and peripheral vascular tone via H(1) receptors in the RVLM and homeostasis on body temperature.

Comparison of source localization of interictal epileptic spike potentials in patients estimated by the dipole tracing method with the focus directly recorded by the depth electrodes.

The purpose of the study was to investigate the accuracy of location of equivalent current dipoles estimated by the dipole tracing method (DT) utilizing a realistic 3-shell (scalp-skull-brain) head model (SSB-DT). Three patients with intractable complex partial seizures, diagnosed as having typical temporal seizures were investigated. We recorded the interictal spike potentials with surface electrodes (International 10/20 system) and with intracerebral depth electrodes simultaneously. We compared the location of dipoles of the spikes estimated by the SSB-DT with the focus of the spikes determined by the recording from the depth electrodes. We found that the location of the dipoles estimated by SSB-DT corresponded to the location of the depth electrodes, which could record the epileptic spikes. This finding proved that SSB-DT is reliable and valid for estimating neural activity in deep locations such as the limbic system.

Involvement of central histaminergic neurons in polypnea induced by hyperthermia in rabbits.

A role of central histamine in the preoptic area/anterior hypothalamus (POA/AH) for the regulation of hyperthermia-induced polypnea was examined in anesthetized, paralyzed, vagotomized and artificially ventilated rabbits. Phrenic nerve activities were recorded to monitor respiratory neuronal output. Hyperthermia increased respiratory frequency by reductions of inspiratory time (T(I)) and expiratory time (T(E)). Pyrilamine, an H1 receptor antagonist, which was applied to the POA/AH reduced polypnea under hyperthermia. The effect of S+alpha-fluoromethylhistidine, a specific inhibitor of histidine decarboxylase, applied in a lateral ventricle was comparable to the effect of pyrilamine on polypnea. Moreover, histamine dihydrochloride applied into the POA/AH at a normal body temperature produced polypnea by reductions of T(I) and T(E). The results suggest that central histamine in the POA/AH contributes to the generation of polypnea in hyperthermia through H1 receptors.

Intervention of thymus and activation-regulated chemokine attenuates the development of allergic airway inflammation and hyperresponsiveness in mice.

Thymus- and activation-regulated chemokine (TARC; CCL17) is a lymphocyte-directed CC chemokine that specifically chemoattracts CC chemokine receptor 4-positive (CCR4(+)) Th2 cells. To establish the pathophysiological roles of TARC in vivo, we investigated here whether an mAb against TARC could inhibit the induction of asthmatic reaction in mice elicited by OVA. TARC was constitutively expressed in the lung and was up-regulated in allergic inflammation. The specific Ab against TARC attenuated OVA-induced airway eosinophilia and diminished the degree of airway hyperresponsiveness with a concomitant decrease in Th2 cytokine levels. Our results for the first time indicate that TARC is a pivotal chemokine for the development of Th2-dominated experimental allergen-induced asthma with eosinophilia and AHR. This study also represents the first success in controlling Th2 cytokine production in vivo by targeting a chemokine.

The effect of anticipatory anxiety on breathing and metabolism in humans.

Respiratory patterns are influenced by cortical and limbic factors and generated by a complex interaction between metabolic requirements and their behavioral effects. Our previous results showed that the temporal pole and the amygdala in the limbic system are related to anxiety and associated with an increase of respiratory frequency, especially in high trait anxiety subjects. The purpose of this study was to investigate the relationship between respiratory patterns and metabolic output during the production of anticipatory anxiety. In all subjects, fR increased without changes in V(O(2)), V(CO(2)) and HR; and PET(CO(2)) decreased during anticipatory anxiety. In the subjects with high trait anxiety, the increase of fR and the decrease of TE were larger than those in the subjects with low trait anxiety. These results suggest that an increase in respiratory frequency is not related to metabolic factors and is consistent with a mechanism involving the limbic system modulating respiratory drive.

Thixotropy of rib cage respiratory muscles in normal subjects.

In this study, we searched for signs of thixotropic behavior in human rib cage respiratory muscles. If rib cage respiratory muscles possess thixotropic properties similar to those seen in other skeletal muscles in animals and humans, we expect resting rib cage circumference would be temporarily changed after deep rib cage inflations or deflations and that these aftereffects would be particularly pronounced in trials that combine conditioning deep inflations or deflations with forceful isometric contractions of the respiratory muscles. We used induction plethysmography to obtain a continuous relative measure of rib cage circumference changes during quiet breathing in 12 healthy subjects. Rib cage position at the end of the expiratory phase (EEP) was used as an index of resting rib cage circumference. Comparisons were made between EEP values of five spontaneous breaths immediately before and after six types of conditioning maneuvers: deep inspiration (DI); deep expiration (DE); DI combined with forceful effort to inspire (FII) or expire (FEI); and DE combined with forceful effort to inspire (FIE) or expire (FEE), both with temporary airway occlusion. The aftereffects of the conditioning maneuvers on EEP values were consistent with the supposition that human respiratory muscles possess thixotropic properties. EEP values were significantly enhanced after all conditioning maneuvers involving DI, and the aftereffects were particularly pronounced in the FII and FEI trials. In contrast, EEP values were reduced after DE maneuvers. The aftereffects were statistically significant for the FEE and FIE, but not DE, trials. It is suggested that respiratory muscle thixotropy may contribute to the pulmonary hyperinflation seen in patients with chronic obstructive pulmonary disease.

Central histamine contributed to temperature-induced polypnea in mice.

Breathing pattern is influenced by body temperature. However, the central mechanism for changing breathing patterns is unknown. Central histamine is involved in heat loss mechanisms in behavioral studies, but little is known about its effect on breathing patterns. We examined first the effect of body temperature on breathing patterns with increasing hypercapnia in conscious mice and then that of the depletion of central histamine by S(+)-alpha-fluoromethylhistidine hydrochloride (alpha-FMH) (100 mg/kg ip), a specific inhibitor of histidine decarboxylase, at normal and raised body temperatures. A raised body temperature increased respiratory frequency with reductions in both inspiratory and expiratory time and decreased tidal volume. On the other hand, alpha-FMH lowered respiratory frequency with a prolongation of expiratory time at the raised temperature; however, this was not observed at a normal temperature. These results indicate that central histamine contributes to an increase in respiratory frequency as a result of a reduction in expiratory time when body temperature is raised.

Lack of temperature-induced polypnea in histamine H1 receptor-deficient mice.

Breathing patterns are influenced by body temperature. However, the central mechanism for changes of breathing patterns is unknown. We previously showed that central histamine contributed to temperature-induced polypnea in mice (Izumizaki, M., Iwase, M., Homma, I., Yanai, K., Watanabe, T. and Watanabe, T., Central histamine contributed to the temperature-induced polypnea in mice, Neurosci. Res., 23 (1999) S282). In this study we examined the role of central histamine H1 receptors in temperature-induced polypnea using wild and mutant mice lacking histamine H1 receptors. Breathing patterns were characterized at two different body temperatures during hypercapnia under conscious conditions. In wild mice a raised body temperature increased respiratory frequency mainly due to a reduction in expiratory time, whereas in mutant mice respiratory frequency did not increase even though the body temperature was elevated. These results indicate that central histamine contributes to an increase in respiratory frequency due to a reduction in expiratory time through histamine H1 receptors when body temperature is raised.

The source generator of respiratory-related anxiety potential in the human brain.

In this study, we used the dipole tracing method of a scalp-skull-brain head model to determine the location of the electric current source which correlates with the increased respiratory rate due to anxiety in humans. Anticipatory anxiety was produced by giving electrical stimulation to the left forefinger after the warning red light. While administering anticipatory anxiety, subjects' anxiety state and respiratory rates increased. In averaged electroencephalogram, which was triggered by onset of inspiration, positive waves were observed approximately 350 ms after the onset of inspiration. In this period of time, dipoles were concentrated in the right temporal pole, and the temporal pole and the amygdala in the most anxious subject. This data suggests that there are respiratory-related neural activities in limbic and paralimbic areas which may correlate with anxiety in humans.

Movement related cortical source for elbow flexion in patients with branchial plexus injury after intercostal-musculocutaneous nerve crossing.

Nine patients with brachial plexus injury whose transected musculo-cutaneous nerves had been sutured with intercostal nerves were examined and the relationship between flexion of the operated elbow and the respiratory movement were shown. Three out of nine patients showed independent control of movement from respiration after regeneration. The primary motor cortex for either flexion of the elbow to the operated side or brisk voluntary inspiration was estimated in the medial vertex region of the frontal cortex by the dipole-tracing method in these three patients. The present results suggest that patients contract the biceps muscle reinnervated by the intercostal nerve independently from respiratory movements using the same primary motor cortex with trunk movements. Functional plasticity may occur in the patients using the primary trunk motor cortex for elbow flexion.

Expiratory time determined by individual anxiety levels in humans.

We have previously found that individual anxiety levels influence respiratory rates in physical load and mental stress (Y. Masaoka and I. Homma. Int. J. Psychophysiol. 27: 153-159, 1997). On the basis of that study, in the present study we investigated the metabolic outputs during tests and analyzed the respiratory timing relationship between inspiration and expiration, taking into account individual anxiety levels. Disregarding anxiety levels, there were correlations between O2 consumption (VO2) and minute ventilation (VE) and between VO2 and tidal volume in the physical load test, but no correlations were observed in the noxious audio stimulation test. There was a volume-based increase in respiratory patterns in physical load; however, VE increased not only for the adjustment of metabolic needs but also for individual mental factors; anxiety participated in this increase. In the high-anxiety group, the VE-to-VO2 ratio, indicating ventilatory efficiency, increased in both tests. In the high-anxiety group, increases in respiratory rate contributed to a VE increase, and there were negative correlations between expiratory time and anxiety scores in both tests. In an awake state, the higher neural structure may dominantly affect the mechanism of respiratory rhythm generation. We focus on the relationship between expiratory time and anxiety and show diagrams of respiratory output, allowing for individual personality.

Respiratory network function in the isolated brainstem-spinal cord of newborn rats.

The in vitro brainstem-spinal cord preparation of newborn rats is an established model for the analysis of respiratory network functions. Respiratory activity is generated by interneurons, bilaterally distributed in the ventrolateral medulla. In particular non-NMDA type glutamate receptors constitute excitatory synaptic connectivity between respiratory neurons. Respiratory activity is modulated by a diversity of neuroactive substances such as serotonin, adenosine or norepinephrine. Cl(-)-mediated IPSPs provide a characteristic pattern of membrane potential fluctuations and elevation of the interstitial concentration of (endogenous) GABA or glycine leads to hyperpolarisation-related suppression of respiratory activity. Respiratory rhythm is not blocked upon inhibition of IPSPs with bicuculline, strychnine and saclofen. This indicates that GABA- and glycine-mediated mutual synaptic inhibition is not crucial for in vitro respiratory activity. The primary oscillatory activity is generated by neurons of a respiratory rhythm generator. In these cells, a set of intrinsic conductances such as P-type Ca2+ channels, persistent Na+ channels and G(i/o) protein-coupled K+ conductances mediates conditional bursting. The respiratory rhythm generator shapes the activity of an inspiratory pattern generator that provides the motor output recorded from cranial and spinal nerve rootlets in the preparation. Burst activity appears to be maintained by an excitatory drive due to tonic synaptic activity in concert with chemostimulation by H+. Evoked anoxia leads to a sustained decrease of respiratory frequency, related to K+ channel-mediated hyperpolarisation, whereas opiates or prostaglandins cause longlasting apnea due to a fall of cellular cAMP. The latter observations show that this in vitro model is also suited for analysis of clinically relevant disturbances of respiratory network function.

In-phase chest wall vibration decreases dyspnea during arm elevation in chronic obstructive pulmonary disease patients.

In-phase chest wall vibration (IPV) is known to decrease dyspnea in patients with chronic obstructive pulmonary disease (COPD) at rest and during leg exercise. In the present study, the effects of IPV (100 Hz) on dyspnea and arm fatigue during upper extremity activity were studied in 9 patients with COPD (mean FEV1, 0.95 l). Dyspnea and arm fatigue (modified Borg scale) and ventilatory variables were measured during arm elevation (AE) with weights lifted straight above the head with and without IPV. Mean dyspnea during AE was 3.3 without IPV and 2.1 with IPV (p<0.05), but, arm fatigue, oxygen saturation and end-tidal FCO2 were not affected by IPV. Minute ventilation during AE was significantly increased with IPV in 5 of 9 patients. The results suggest that IPV decreases dyspnea during AE.

Central histaminergic neurons regulate rabbit tracheal tension through the cervical sympathetic nerve.

We previously showed that stimulation of the posterior hypothalamus decreases tracheal tension and involves central histaminergic neurons. In the present study, we reveal that central histaminergic neurons project to the rostral ventrolateral medulla and affect cervical sympathetic nervous activity in rabbits. Administration of histamine into the fourth ventricle increased cervical sympathetic nervous activity and decreased tracheal tension. These effects were inhibited by administration of a histamine H receptor antagonist, pyrilamine, into the fourth ventricle. Unilateral injection of DL-homocysteic acid into the tuberomammillary nucleus increased cervical sympathetic nervous activity, an effect was antagonized by bilateral injection of pyrilamine into the rostral ventrolateral medulla. The pulse correlogram between the stimulation pulse applied to the tuberomammillary nucleus and the cervical sympathetic nerve activity showed a mode at 150 to 200 ms, which was reduced by pyrilamine administration into the fourth ventricle. Fibers anterogradely labeled by Phaseolus vulgaris leucoagglutinin (PHA-L) injected into the tuberomammillary nucleus were distributed in the A1, A2, C1, and C2 areas which are determined by tyrosine hydroxylase-immunohistochemistry. PHA-L positive neurons were in close contact with tyrosine hydroxylase-immunoreactive neurons in these four areas. Cell bodies in the tuberomammillary nucleus retrogradely labeled with fluorogold from the rostral ventrolateral medulla were immunoreactive with histamine. These results suggest that an excitatory efferent pathway projects from the tuberomammillary nucleus to the cervical sympathetic nerve and that the histaminergic neurons of this pathway influence tracheal tension through the rostral ventrolateral medulla.