The effects of doxapram on medullary respiratory neurones in brainstem-spinal cord preparations from newborn rats.

Doxapram is the only dedicated respiratory stimulant used to aid recovery of breathing after major surgery. Doxapram acts on peripheral chemoreceptors and although the central action of doxapram has been suggested, its detailed neuronal mechanism is unknown. We assessed doxapram-induced changes in spontaneous cervical nerve (C4) inspiratory activity and the firing of action potentials in pre-inspiratory and inspiratory neurones in the medulla. Experiments were performed in neonatal rat brainstem-spinal cord preparations, which can produce respiratory rhythm for several hours under in vitro conditions. Doxapram application (for 15 min) increased the frequency and amplitude of C4 activity dose-dependently. Doxapram induced changes in the electrophysiological properties of pre-inspiratory and inspiratory neurones. Our results suggest that respiratory activity enhancement was likely to be induced via effects on the potassium channels of pre-inspiratory and inspiratory neurones and indicate the central actions of doxapram.

Rnx deficiency results in congenital central hypoventilation.

The genes Tlx1 (Hox11), Enx (Hox11L2, Tlx-2) and Rnx (Hox11L2, Tlx-3) constitute a family of orphan homeobox genes. In situ hybridization has revealed considerable overlap in their expression within the nervous system, but Rnx is singularly expressed in the developing dorsal and ventral region of the medulla oblongata. Tlx1-deficient and Enx-deficient mice display phenotypes in tissues where the mutated gene is singularly expressed, resulting in asplenogenesis and hyperganglionic megacolon, respectively. To determine the developmental role of Rnx, we disrupted the locus in mouse embryonic stem (ES) cells. Rnx deficient mice developed to term, but all died within 24 hours after birth from a central respiratory failure. The electromyographic activity of intercostal muscles coupled with the C4 ventral root activity assessed in a medulla-spinal cord preparation revealed a high respiratory rate with short inspiratory duration and frequent apnea. Furthermore, a coordinate pattern existed between the abnormal activity of inspiratory neurons in the ventrolateral medulla and C4 motorneuron output, indicating a central respiratory defect in Rnx mice. Thus, Rnx is critical for the development of the ventral medullary respiratory centre and its deficiency results in a syndrome resembling congenital central hypoventilation.

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.

Opioids prolong and anoxia shortens delay between onset of preinspiratory (pFRG) and inspiratory (preBötC) network bursting in newborn rat brainstems.

Differential responses to opioids established the hypothesis that pre/postinspiratory (Pre-I) neurons of the parafacial respiratory group (pFRG) and inspiratory (Insp) neurons of the pre-Bötzinger complex (preBötC) constitute a dual brainstem respiratory center. For further analysis of pFRG/preBötC interactions, we studied in newborn rat brainstem-spinal cord preparations opioid and anoxia effects on histologically identified pFRG-driven "type-I" Insp preBötC neurons and Pre-I neurons from three distinct respiratory brainstem regions. The micro-opioid [D-Ala(2),N-Me-Phe(4),Gly(5)-ol]-enkephalin (DAMGO) slowed inspiratory-related cervical nerve bursts quantally, whereas anoxia induced nonquantal slowing and repetitive cervical bursts. DAMGO had no effect on membrane potential or input resistance of Pre-I neurons, while anoxia hyperpolarized them (approximately 5 mV) and decreased their resistance (approximately 30%). DAMGO prolonged the preinspiratory phase of Pre-I neuron bursting, whereas anoxia caused a shift to postinspiratory (48%) or inspiratory (22%) activity and silenced further 30% of cells. Pre-I neuron responses were not correlated with their rostrocaudal location or morphology. Neither DAMGO nor anoxia changed membrane potential of type-I neurons, but decreased their input resistance by 33% and 21%, respectively. The opposite DAMGO- and anoxia-evoked phase shifts of Pre-I neuron activity were reflected by corresponding shifts of pre/postinspiratory drive potentials in type-I neurons and, partly, by voltage-sensitive dye-imaged medullary neuronal population activities. The findings suggest that opioids presynaptically delay activation of type-I neurons as the target of drive from the pFRG to the preBötC. Contrary, anoxia seems to partly synchronize the pFRG and preBötC rhythm generators. This may enhance inspiratory and postinspiratory medullary activities for triggering multiple inspiratory motor bursts.

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.

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.

Distribution of respiration-related neuronal activity in the thoracic spinal cord of the neonatal rat: An optical imaging study.

The inspiratory motor outputs are larger in the intercostal muscles positioned at more rostral segments. To obtain further insights into the involvement of the spinal interneurons in the generation of this rostrocaudal gradient, the respiratory-related neuronal activities were optically recorded from various thoracic segments in brainstem-spinal cord preparations from 0- to 2-day-old rats. The preparation was stained with a voltage-sensitive dye, and the optical signals from about 2.5s before to about 7.7s after the peak of the C4 inspiratory discharge were obtained. Respiratory-related depolarizing signals were detectable from the ventral surface of all thoracic segments. Since the local blockage of the synaptic transmission in the thoracic spinal cord induced by the low-Ca(2+) superfusate blocked all respiratory signals, it is likely that these signals came from spinal neurons. Under the-low Ca(2+) superfusate, ventral root stimulation, inducing antidromic activation of motoneurons, evoked depolarizing optical signals in a restricted middle area between the lateral edge and midline of the spinal cord. These areas were referred to as 'motoneuron areas'. The respiratory signals were observed not only in the motoneuron areas but also in areas medial to the motoneuron areas, where interneurons should exist; these were referred to as 'interneuron areas'. The upper thoracic segments showed significantly larger inspiratory-related signals than the lower thoracic segments in both the motoneuron and interneuron areas. These results suggest that the inspiratory interneurons in the thoracic spinal cord play a role in the generation of the rostrocaudal gradient in the inspiratory intercostal muscle activity.

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.

Localization and properties of respiratory neurons in the rostral pons of the newborn rat.

The distribution and discharge pattern of respiratory neurons in the 'pneumotaxic center' of the rostral pons in the rat has remained unknown. We performed optical recordings and whole-cell patch clamp recordings to clarify respiratory neuron activity in the rostral pons of a brainstem-spinal cord preparation from a newborn rat. Inspiratory nerve activity was recorded in the 4th cervical nerve and used as a trigger signal for optical recordings. Respiratory neuron activity was detected in the limited region of the rostral-lateral pons. The main active region was presumed to be primarily the Kölliker-Fuse nucleus. The location of respiratory neurons was further confirmed by Lucifer Yellow staining after conducting whole-cell recordings. From a membrane potential analysis of the respiratory neurons in the rostral pons, the respiratory neurons were divided into four types: inspiratory neuron (71.9%), pre-inspiratory neuron (5.3%), post-inspiratory neuron (19.3%), and expiratory neuron (3.5%). A noticeable difference between pontine and medullary respiratory neurons was that post-inspiratory neurons were more frequently encountered in the pons. Application of a mu-opioid agonist, [d-Ala2, N-Me-Phe4, Gly5-ol]-enkephalin, transformed the burst pattern of post-inspiratory neurons into that of pre-inspiratory neurons. The electrical stimulation of the sensory root of the trigeminal nerve induced three types of responses in 85% of pontine respiratory neurons: inhibitory postsynaptic potentials (42.7%), excitatory postsynaptic potentials (37.7%) and no response (15.1%). Our findings provide the first evidence in the rat for the presence of respiratory neurons in the rostral pons, with localization in the lateral region approximately overlapping with the Kölliker-Fuse nucleus.

Effects of ouabain on respiratory rhythm generation in brainstem-spinal cord preparation from newborn rats and in decerebrate and arterially perfused in situ preparation from juvenile rats.

The significance of Na/K-ATPase on respiratory rhythm generation is not well understood. We investigated the effects of the Na/K-ATPase blocker, ouabain, on respiratory rhythm. Experiments were performed with brainstem-spinal cord preparation from 0 to 3-day-old Wistar rats and with decerebrate and arterially perfused in situ preparation from juvenile rats (postnatal day 11-13). Newborn rat preparations were superfused at a rate of 3.0 ml/min with artificial cerebrospinal fluid, equilibrated with 95% O2 and 5% CO2, pH 7.4, at 26-27 °C. Inspiratory activity was monitored from the fourth cervical ventral root (C4). Application of ouabain (15-20 min) resulted in a dose-dependent increase in the burst rate of C4 inspiratory activity. After washout, the burst rate further increased to reach quasi-maximum values under each condition (e.g. 183% of control in 1 µM, 253% in 10 µM, and 303% in 20 µM at 30 min washout). Inspiratory or pre-inspiratory neurons in the rostral ventrolateral medulla were depolarized. We obtained similar results (i.e. increased phrenic burst rate) in an in situ perfused preparation of juvenile rats. Genes encoding the Na/K-ATPase α subunit were expressed in the region of the parafacial respiratory group (pFRG) in neonatal rats, suggesting that cells (neurons and/or glias) in the pFRG were one of the targets of ouabain. We concluded that Na/K-ATPase activity could be an important factor in respiratory rhythm modulation.

Uric acid, indoxyl sulfate, and methylguanidine activate bulbospinal neurons in the RVLM via their specific transporters and by producing oxidative stress.

Patients with chronic renal failure often have hypertension, but the cause of hypertension, other than an excess of body fluid, is not well known. We hypothesized that the bulbospinal neurons in the rostral ventrolateral medulla (RVLM) are stimulated by uremic toxins in patients with chronic renal failure. To investigate whether RVLM neurons are sensitive to uremic toxins, such as uric acid, indoxyl sulfate, or methylguanidine, we examined changes in the membrane potentials (MPs) of bulbospinal RVLM neurons of Wister rats using the whole-cell patch-clamp technique during superfusion with these toxins. A brainstem-spinal cord preparation that preserved the sympathetic nervous system was used for the experiments. During uric acid, indoxyl sulfate, or methylguanidine superfusion, almost all the RVLM neurons were depolarized. To examine the transporters for these toxins on RVLM neurons, histological examinations were performed. The uric acid-, indoxyl sulfate-, and methylguanidine-depolarized RVLM neurons showed the presence of urate transporter 1 (URAT 1), organic anion transporter (OAT)1 or OAT3, and organic cation transporter (OCT)3, respectively. Furthermore, the toxin-induced activities of the RVLM neurons were suppressed by the addition of an anti-oxidation drug (VAS2870, an NAD(P)H oxidase inhibitor), and a histological examination revealed the presence of NAD(P)H oxidase (nox)2 and nox4 in these RVLM neurons. The present results show that uric acid, indoxyl sulfate, and methylguanidine directly stimulate bulbospinal RVLM neurons via specific transporters on these neurons and by producing oxidative stress. These uremic toxins may cause hypertension by activating RVLM neurons.

Possible synaptic connections of expiratory neurons in the medulla of newborn rat in vitro.

To elucidate synaptic interactions between expiratory (Exp) and other respiratory neurons in the ventrolateral medulla of brain stem-spinal cord preparations from newborn rats, we analyzed postsynaptic potentials in Exp and a subclass of inspiratory (Insp) neurons using whole-cell recordings. About 72% of the Exp neurons (Exp-p-i) showed Cl--dependent synaptic inhibition during the Insp and pre/post-Insp phases, corresponding to the active phase of the Insp and pre-inspiratory (Pre-I) neurons, respectively. The other 28% neurons (Exp-i) received Cl--dependent inhibition during the Insp phase only. Some Insp neurons showed reversed IPSPs during the active phase of Exp-p-i after Cl-loading. We suggest the existence of inhibitory connections from Pre-I and Insp to Exp and from Exp to Insp neurons. Basic synaptic connections among respiratory neurons similar to those in intact adult mammals may already exist in newborn rats.

Studies of the respiratory center using isolated brainstem-spinal cord preparations.

It has been ten years since a brainstem-spinal cord preparation isolated from a newborn rat was introduced for study of the mammalian respiratory center. Here, I briefly summarize first, these studies, which include the tissue condition of in vitro preparations, respiratory reflexes, pharmacology, rhythm generation, respiratory chemoreception, phrenic motoneurons, regulation from pons, and development of a respiratory center. In the latter half of this paper, I focus on the neural mechanisms of respiratory rhythm generation. A current hypothesis for the central pattern generator of respiration proposed by the author's group is that the respiratory rhythm generator, composed of pre-inspiratory neurons in the rostral ventrolateral medulla, produces the primary rhythm of respiration and triggers an inspiratory pattern generator composed of inspiratory neurons in the rostral and the caudal ventrolateral medulla.

Optical imaging of respiratory burst activity in newborn rat medullary block preparations.

We report on the optical imaging of excitation propagation induced by electrical stimulation of the nucleus tractus solitarius (NTS) area and subsequent inspiratory burst activity in the ventrolateral medulla (VLM) of a medullary block preparation. A medullary block preparation with a thickness of 1.0-1.4 mm was made from brainstems isolated from 0- to 4-day-old rats and stained with a fluorescent voltage-sensitive dye, RH795. Neuronal responses in the VLM evoked by electrical stimulation were recorded as a fluorescence change using an optical recording apparatus with a 128 x 128 photodiode array and a maximum time resolution of 0.6 ms. Motoneuronal activity was simultaneously recorded at the contralateral hypoglossal nerve roots. Neuronal excitation evoked by stimulation of the NTS area propagated to the VLM through the intermediate reticular zone (IRt). In contrast, caudal VLM stimulation induced excitation which propagated to the rostral VLM without any detectable excitation propagation in the IRt toward the NTS area from the VLM. NTS stimulation also induced an inspiratory burst activity in the hypoglossal nerve root activity with a 150-200 ms delay. Fluorescence changes corresponding to the inspiratory burst activity were observed in the VLM which coincided with the area in which the localization of many respiratory neurons had been demonstrated in previous studies using whole-brainstem preparations. The present results show the feasibility of using optical recordings for the analysis of respiratory neuron activity as well as for analysis of the transmission pathway of afferent and/or efferent information in the medulla.

Respiratory rhythm generator neurons in medulla of brainstem-spinal cord preparation from newborn rat.

Rhythmic neuronal activity preceding C4 inspiratory activity (Pre-I neuron activity) was recorded in rostral ventrolateral (near ventral surface) medulla of brainstem-spinal cord preparation isolated from newborn rat. Vagal stimulation inhibited C4 activity but not Pre-I neuron activity. Rhythmic Pre-I neuron-like activity was still recorded in the block of rostral medulla after transection. Results suggest that Pre-I neurons generate the basic respiratory rhythm and trigger inspiratory activity.

Primary respiratory rhythm generator in the medulla of brainstem-spinal cord preparation from newborn rat.

It has been previously demonstrated that rhythmically firing neurons (Pre-I neurons) preceding cervical root (C4 or C5) inspiratory activity, localized in the rostral ventrolateral medulla (RVL), are important in the generation of the basic respiratory rhythm in brainstem-spinal cord preparations from newborn rats. We examined the effects of single and continuous electrical stimulation applied to the RVL on Pre-I and C4 activities in these preparations. We verified that the phase of respiratory rhythm was reset when Pre-I firing was induced in both right and left RVL by single shock stimulation, whether C4 activity appeared or not. Lower frequency and intensity of continuous pulse train stimulation in the RVL enhanced Pre-I activity, and hence C4 activity, whereas higher frequency and intensity inhibited both. The results suggest that synchronous burst activity between the right and left Pre-I neurons must be above a certain level (in its intraburst firing rate) to trigger C4 inspiratory activity and, therefore, that cooperation among Pre-I neurons is important for induction of rhythmic inspiratory drive. After bilateral lesions of the caudal ventrolateral medulla, Pre-I neurons retained their rhythmic activity, while C4 activity disappeared. Present results further confirmed our hypothesis that Pre-I neurons are the primary generator of respiratory rhythm. We propose a hypothetical model of the generation of rhythmic respiratory activity.

Effect of substance P on respiratory rhythm and pre-inspiratory neurons in the ventrolateral structure of rostral medulla oblongata: an in vitro study.

The pre-inspiratory (Pre-I) neurons which fire in the pre- and usually also during the post-inspiratory phase are located in the ventrolateral structures of the rostral medulla. They are suggested as primary rhythm generating neurons for respiration. These have been studied in isolated brainstem-spinal cord preparations from newborn 0-5-day-old rats. We have found that application of substance P (SP) enhanced the respiratory rhythm as measured by C4 ventral root and pre-I neuronal activities. Furthermore, the effect of SP was dependent on basal respiratory rate. An increase of the Pre-I and C4 burst rate by SP was clearer when the basal respiratory rhythm was somewhat lower. Moreover, long lasting depression of respiratory rate after the application of the alpha 2-agonist clonidine was reversed by SP. On the other hand, an inhibitory effect appeared in preparations with a higher basal respiratory rate, while the Pre-I burst rate tended to increase during SP perfusion. During chemical synaptic transmission blockade by perfusion with low Ca2+, high Mg2+ solution, a pre-I burst retained or completely blocked was found to be enhanced or reactivated by SP perfusion. The results suggest a direct postsynaptic action of SP, which could strongly stimulate burst generating properties of Pre-I neurons.

Effects of cAMP on respiratory rhythm generation in brainstem-spinal cord preparation from newborn rat.

Involvement of cAMP in the generation of respiratory rhythm was studied in newborn rat brainstem-spinal cord preparations. The respiratory rhythm was monitored by C4 inspiratory activity and/or pre-inspiratory (Pre-I) activity of neurons in the rostral ventrolateral medulla; previously suggested to be primary rhythm generating neurons which have pacemaker properties. The effects of four cAMP-increasing agents (forskolin, IBMX, Db-cAMP, and 8-Br-cAMP) on this neuronal activity were examined. Perfusion with forskolin (3-10 microM) increased the burst rate of C4 inspiratory activity in 20 of 32 preparations, but in 8 of those the increase was preceded by transient depression. The facilitation of the respiratory rhythm was greater whenever the burst rate before forskolin treatment was lower. The Pre-I neuron burst rate, which was recorded together with C4 activity, predominantly increased with forskolin treatment. The effects of IBMX, Db-cAMP and 8-Br-cAMP were similar to those of forskolin, but they were slightly less potent. Long-lasting depression of the respiratory rhythm (C4 and Pre-I activity) by clonidine, which might decrease intracellular cAMP level via alpha 2-receptors, was reversed by forskolin. To investigate the direct effects of the cAMP-increasing agents on Pre-I neurons, Pre-I activity was isolated by blocking the chemical synaptic transmission by incubation in a low Ca solution (0.2 mM Ca2+, 5 mM Mg2+). Forskolin (5-10 microM), IBMX (5-10 microM), Db-cAMP (0.2-0.4 mM), and 8-Br-cAMP (0.4-0.75 mM) all enhanced the burst rate of isolated Pre-I neurons.(ABSTRACT TRUNCATED AT 250 WORDS)

Localization of respiratory rhythm-generating neurons in the medulla of brainstem-spinal cord preparations from newborn rats.

We describe the location of Pre-I neurons, which are important to respiratory rhythm generation, in the rostral medulla of brainstem-spinal cord preparations isolated from newborn rats. This neuronal group was delimited in the reticular formation slightly medial to the caudal area of the facial nucleus and near the ventral surface. The effects of electrical stimulation and lesions in that region were also examined with respect to respiratory rhythm generation. Single shock stimulation induced Pre-I neuron firing and reset the phase of the respiratory rhythm. Electrolytic lesions in the Pre-I neuron region reduced the respiratory rate.

Effects of histamine inhalation and airway vibration on phrenic nerve activity in rabbits.

Experiments were performed on 8 rabbits under urethane-chloralose anesthesia. Histamine aerosol (5%) administered into the trachea by a cannula produced tonic activity of the phrenic nerve, with discharges also being evident during the expiratory phase. Airway vibration (100 Hz) inhibited this tonic activity and caused silence during the expiratory phase.