Hiroshi Onimaru

A novel functional neuron group for respiratory rhythm generation in the ventral medulla.

We visualized respiratory neuron activity covering the entire ventral medulla using optical recordings in a newborn rat brainstem–spinal cord preparation stained with voltage-sensitive dye. We measured optical signals from several seconds before to several seconds after the inspiratory phase using the inspiratory motor nerve discharge as the trigger signal; we averaged the optical signals of 50–150 respiratory cycles to obtain an optical image correlating particularly to inspiratory activity. The optical images we obtained from the ventral approach indicated that neuron activity first appeared during the respiratory cycle in the limited region of the rostral ventrolateral medulla (RVLM), preceding the onset of inspiratory activity by ∼500 msec. During the inspiratory phase, plateau activity appeared in the more caudal ventrolateral medulla at the level of the most rostral roots of the XIIth nerve. Comparison with electrophysiological recordings from respiratory neurons in the RVLM suggested that the optical signals preceding the inspiratory burst reflect preinspiratory neuron activity in this area. This RVLM area was determined to be ventrolateral to the facial nucleus and close to the ventral surface. We referred to this functional neuron group as the para-facial respiratory group (pFRG). Partial, bilateral electrical lesioning of the pFRG significantly reduced the respiratory frequency, together with changes in the spatiotemporal pattern of respiratory neuron activity. Our findings suggest that the pFRG comprises a neuronal population that is involved in the primary respiratory rhythm generation in the rostrocaudally extending respiratory neuron network of the medulla.

Tlx3 and Tlx1 are post-mitotic selector genes determining glutamatergic over GABAergic cell fates

Glutamatergic and GABAergic neurons mediate much of the excitatory and inhibitory neurotransmission, respectively, in the vertebrate nervous system. The process by which developing neurons select between these two cell fates is poorly understood. Here we show that the homeobox genes Tlx3 and Tlx1 determine excitatory over inhibitory cell fates in the mouse dorsal spinal cord. First, we found that Tlx3 was required for specification of, and expressed in, glutamatergic neurons. Both generic and region-specific glutamatergic markers, including VGLUT2 and the AMPA receptor Gria2, were absent in Tlx mutant dorsal horn. Second, spinal GABAergic markers were derepressed in Tlx mutants, including Pax2 that is necessary for GABAergic differentiation, Gad1/2 and Viaat that regulate GABA synthesis and transport, and the kainate receptors Grik2/3. Third, ectopic expression of Tlx3 was sufficient to suppress GABAergic differentiation and induce formation of glutamatergic neurons. Finally, excess GABA-mediated inhibition caused dysfunction of central respiratory circuits in Tlx3 mutant mice.

Opioid-resistant respiratory pathway from the preinspiratory neurones to abdominal muscles: in vivo and in vitro study in the newborn rat

We report that after spontaneous breathing movements are stopped by administration of opioids (opioid‐induced apnoea) in neonatal rats, abdominal muscles continue to contract at a rate similar to that observed during periods of ventilation. Correspondingly, in vitro bath application of a μ opioid receptor agonist suppresses the activity of the fourth cervical root (C4) supplying the diaphragm, but not the rhythmic activity of the first lumbar root (L1) innervating the abdominal muscles. This indicates the existence of opioid‐resistant rhythmogenic neurones and a neuronal pathway transmitting their activity to the abdominal motoneurones. We have investigated this pathway by using a brainstem‐spinal cord preparation of the neonatal rat. We identified bulbospinal neurones with a firing pattern identical to that of the L1 root. These neurones were located caudal to the obex in the vicinity of the nucleus retroambiguus. Resting potentials ranged from ‐49 to ‐40 mV (mean ±s.d. ‐44.0 ± 4.3 mV). The mean input resistance was 315.5 ± 54.8 MΩ. The mean antidromic latency from the L1 level was 42.8 ± 4.4 ms. Axons crossed the midline at the level of the cell body. The activity pattern of the bulbospinal neurones and the L1 root consisted of two bursts per respiratory cycle with a silent period during inspiration. This pattern is characteristic of preinspiratory neurones. We found that 11 % of the preinspiratory neurones projected to the area where the bulbospinal neurones were located. These preinspiratory neurones were found in the rostral ventrolateral medulla close (200‐350 μm) to the ventral surface at the level of the rostral half of the nucleus retrofacialis. Our data suggest the operation of a disynaptic pathway from the preinspiratory neurones to the L1 motoneurones in the in vitro preparation. We propose that the same pathway is responsible for rhythmic activation of the abdominal muscles during opioid‐induced apnoea in the newborn rat.

Rnx deficiency results in congenital central hypoventilation.

The genes Tlx1 (Hox11), Enx (Hox11L1, 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.

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.

Respiratory rhythm generator neurons in medulla of brainstem-spinal cold 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.

Firing properties of respiratory rhythm generating neurons in the absence of synaptic transmission in rat medulla in vitro

SummaryIt has previously been demonstrated that Pre-I neurons, localized in the rostral ventrolateral medulla, are important in the generation of the primary respiratory rhythm in brainstemspinal cord preparations from newborn rats. To investigate whether or not Pre-I neurons have endogenous pacemaker properties, we examined Pre-I neuron activity before and after chemical synaptic transmission was blocked by incubation in a low Ca2+ (0.2 mM), high Mg2+ (5 mM) solution (referred to here as low Ca). After incubation for about 30 min in low Ca, 28 (52%, type-1) out of 54 neurons tested in 27 preparations retained apparent rhythmic (phasic) activity after complete disappearance of C4 inspiratory activity. Sixteen neurons (30%, type-2) fired tonically and 10 (18%, type-3) were silent. We examined the effects of synaptic blockade on 14 inspiratory neurons in the RVL. The firing of all 14 neurons in 9 preparations disappeared concomitantly with the disappearance of C4 activity in low Ca. When the pH of the low Ca solution was lowered with a decrease in NaHCO3 concentration from 7.4 to 7.1, the firing rate of the Pre-I neurons (type-1) increased from 12 to 18/min. In conclusion, the generator of respiratory rhythm in the newborn rat is probably a neuronal network with chemical synapses that functions mainly through the endogenous Pre-I pacemaker cells. Intrinsic chemoreception in the rhythm generator is probably important in frequency control of respiratory rhythm.

CO2-Sensitive Preinspiratory Neurons of the Parafacial Respiratory Group Express Phox2b in the Neonatal Rat

Phox2b protein is a specific marker for neurons in the parafacial region of the ventral medulla, which are proposed to play a role in central chemoreception and postnatal survival. Mutations of PHOX2B cause congenital central hypoventilation syndrome. However, there have been no reports concerning electrophysiological characteristics of these Phox2b-expressing neurons in the parafacial region of the neonate immediately after birth. This region overlaps with the parafacial respiratory group (pFRG) composed predominantly of preinspiratory (Pre-I) neurons that are involved in respiratory rhythm generation. We studied (1) whether pFRG neurons are Phox2b immunoreactive or not and (2) whether they show intrinsic CO2 chemosensitivity. We found that most pFRG/Pre-I neurons were Phox2b immunoreactive and depolarized upon increase in CO2 concentration under condition of action potential-dependent synaptic transmission blockade by tetrodotoxin. We also confirmed that these pFRG neurons expressed neurokinin-1 receptor. They were tyrosine hydroxylase negative and presumed to be glutamatergic. Our findings suggest that Phox2b-expressing parafacial neurons play a role in respiratory rhythm generation as well as central chemoreception and thus are essential for postnatal survival.

Opioid Action on Respiratory Neuron Activity of the Isolated Respiratory Network in Newborn Rats

BackgroundUnderlying mechanisms behind opioid-induced respiratory depression are not fully understood. The authors investigated changes in burst rate, intraburst firing frequency, membrane properties, as well as presynaptic and postsynaptic events of respiratory neurons in the isolated brainstem after administration of opioid receptor agonists. MethodsNewborn rat brainstem–spinal cord preparations were used and superfused with &mgr;-, &kgr;-, and &dgr;-opioid receptor agonists. Whole cell recordings were performed from three major classes of respiratory neurons (inspiratory, preinspiratory, and expiratory). ResultsMu- and &kgr;-opioid receptor agonists reduced the spontaneous burst activity of inspiratory neurons and the C4 nerve activity. Forty-two percent of the inspiratory neurons were hyperpolarized and decreased in membrane resistance during opioid-induced respiratory depression. Furthermore, under synaptic block by tetrodotoxin perfusion, similar changes of inspiratory neuronal membrane properties occurred after application of &mgr;- and &kgr;-opioid receptor agonists. In contrast, resting membrane potential and membrane resistance of preinspiratory and majority of expiratory neurons were unchanged by opioid receptor agonists, even during tetrodotoxin perfusion. Simultaneous recordings of inspiratory and preinspiratory neuronal activities confirmed the selective inhibition of inspiratory neurons caused by &mgr;- and &kgr;-opioid receptor agonists. Application of opioids reduced the slope of rising of excitatory postsynaptic potentials evoked by contralateral medulla stimulation, resulting in a prolongation of the latency of successive first action potential responses. ConclusionsMu- and &kgr;-opioid receptor agonists caused reduction of final motor outputs by mainly inhibiting medullary inspiratory neuron network. This inhibition of inspiratory neurons seems to be a result of both a presynaptic and postsynaptic inhibition. The central respiratory rhythm as reflected by the preinspiratory neuron burst rate was essentially unaltered by the agonists.

Whole cell recordings from respiratory neurons in the medulla of brainstem-spinal cord preparations isolated from newborn rats

In brainstem-spinal cord preparations isolated from newborn rats, a whole cell recording technique was applied to record membrane potentials of inspiratory (Insp) and pre-inspiratory (Pre-I) neurons in the ventrolateral medulla. Labelling of these respiratory neurons with Lucifer Yellow allowed analysis of their locations and morphology. Intracellular membrane potentials from 25 Insp neurons were recorded. Average resting membrane potential was −49 mV (n=25) and input resistance was 306 MΩ. Insp neurons were classified into three types from the patterns of synaptic potentials. Type I neurons (n=11) had a high probability of excitatory postsynaptic potentials (EPSPs) in the pre- and post-inspiratory phases. Type II neurons (n=7) showed abrupt transition to the burst phase from the resting potential level without increased EPSPs in the preinspiratory phase. Type III neurons (n=7) were hyperpolarized by inhibitory postsynaptic potentials (IPSPs) in the pre- and post-inspiratory phases. These Insp neurons, located in the ventrolateral medulla 80–490μm from the ventral surface, were 10–30 μm in diameter, and had various soma shapes (pyramidal, spherical or fusiform). Intracellular membrane potentials from 24 Pre-I neurons were recorded. The average resting membrane potential was −45 mV (n=24), and the input resistance was 320 MΩ. Typical Pre-I neurons showed fairly great depolarization accompanied by action potentials during their burst phase and repolarization during the inspiratory phase. Most Pre-I neurons appeared to have a high level of synaptic activity. These cells were located in the ventrolateral medulla 50–440 μm below the ventral surface and had pyramidal or fusiform somas of 10–25 μm in diameter. Stimulation of the ipsilateral IXth, Xth roots or the spinal cord (C3 level) induced orthodromic responses in most Insp or Pre-I neurons. An antidromic action potential was induced in only one Pre-I neuron by stimulation at the ipsilateral C3 level. Many Insp or Pre-I neurons had dendrites that terminated close to the ventral surface of the medulla. The present study revealed postsynaptic activity of respiratory neurons in the rostral ventrolateral medulla, which is consistent with the excitatory and inhibitory synaptic connections from Pre-I neurons to Insp neurons, and inhibitory synaptic connections for Insp neurons to Pre-I neurons.